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Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Aqueous Spray-dried Green Tea Extract Regulates Body Weight and Epididymal Fat Accumulation in Mice (열수 녹차추출물이 생쥐의 체중 및 부고환 지방축적 조절에 미치는 영향)

  • Park, Pil-Joon;Kim, Chae-Wook;Cho, Si-Young;Rha, Chan-Su;Seo, Dae-Bang;Lee, Sang-Jun
    • Korean Journal of Food Science and Technology
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    • v.42 no.1
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    • pp.103-108
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    • 2010
  • To obtain the best yield of the beneficial ingredients in green tea, such as catechins, green tea powder is most often prepared by ethyl alcohol extraction. However, the taste, cost and composition of ethyl alcohol extract is different from aqueous spray-dried green tea extract (aq-GTE). Specifically, aq-GTE has a better flavor, lower production costs and higher purity when compared to ethyl alcohol extract. In this study, we elucidated the effect of aq-GTE on diet-induced obesity in male C57BL/6J mice following dose-dependent oral administration of aq-GTE. After eight weeks, the body weight was reduced by 13-17% in mice fed 200 mg/kg bw aq-GTE ($12.468{\pm}0.45\;g$; p<0.05) and 20-25% in mice fed 400 mg/kg bw aq-GTE ($11.259{\pm}0.61\;g$; p<0.05) when compared with the high-fat diet (HFD) control group mice ($14.714{\pm}0.95\;g$; p<0.05). The correlation between epididymal fat accumulation and body weight also decreased by approximately 26.6% (p<0.05) in mice fed a HFD with aq-GTE 400 mg/kg bw. Finally, serum parameters such as the triglyceride, glucose and cholesterol levels in the HFD groups were reduced by the aq-GTE 400 mg/kg bw diet. Analysis on glutamic-pyruvic transaminase, blood urea nitrogen and development of hepatic steatosis revealed no histologic evidence of hepatotoxicity in HFD mice fed aq-GTE. Overall, our results imply that aq-GTE is able to regulate body weight and fat accumulation in mice.

Elucidation of Dishes High in N-Nitrosamines Using Total Diet Study Data (총식이조사 자료를 이용한 음식별 니트로사민 함량 분포 규명)

  • Choi, Seul Ki;Lee, Youngwon;Seo, Jung-eun;Park, Jong-eun;Lee, Jee-yeon;Kwon, Hoonjeong
    • Journal of Food Hygiene and Safety
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    • v.33 no.5
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    • pp.361-368
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    • 2018
  • N-nitrosamines are probable or possible human carcinogens, which are produced by the reaction between secondary amines and nitrogen oxide in the acidic environment or by heating. Common risk assessment procedure involves the comparison between exposures expressed in the unit, mg/kg body weight/day and the Health-Based Reference dose expressed in the same unit. This procedure is suitable for the policy decision-making and is considered as inappropriate for the consumers to get information about their dietary decision-making. Therefore, the distributions of NDMA (N-nitrosodimethylamine), NDBA (N-nitrosodibutylamine), the six N-nitrosamines (NDMA, NDBA, NDEA (N-nitrosodiethylamine), NPYR (N-nitrosopyrrolidine), NPIP (N-nitrosopiperidine), and NMOR (N-nitrosomorpholine) in the menus grouped based on the presence of main ingredients and cooking methods were analyzed to generate consumer-friendly information regarding food contaminants. Recipes and intakes were taken from 2014 to 2016 KNHANES (The Korean National Health and Nutrition Examination Survey) and only the data from ages of 7 years or older were used. The contamination data were collected from the 2014~2016 Total Diet Study and all the analysis were performed using R software. Rockfish, eel, anchovy broth and pollock were mainly exposed to N-nitrosamines. In terms of cooking methods, soups and stews appeared to contain the highest amount of N-nitrosamines. Cereals, fruits, and dairy products in the ingredient categories, and rice dishes and rice combined with others in recipe categories had the lowest level exposure to N-nitrosamines. In case of N-nitrosamines, unlike other cooking related food contaminants, boiled dishes such as soups and stews and dishes mainly consisting of fishes and shellfishes had highest level of exposure, showing a large discrepancy with the previous thought of processed meat is the main source of N-nitrosamines.

Molecular Epidemiological Characteristics of Vibrio Parahaemolyticus as Recently wilde-spreaded in Korea (최근 한국에서 유행하는 장염비브리오균의 분자 역학적 특성)

  • 김상숙;이희무;이중복
    • KSBB Journal
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    • v.18 no.6
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    • pp.522-528
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    • 2003
  • The purpose of this study is to inquire into molecular epidemiological characteristics of Vibrio parahaemolyticus. For this study, 120 strains(120 strains of Vibrio parahaemolyticus sampled from diarrhea patients) were examined and analyzed for biochemical characteristics, TDH (thermostable direct hemolysin) antibiotics sensitivity and detection of toxR, gyrE, tdh, and tds gents. G-S PCR (Group Specific Polymerase), PFGE (Pulsed-field Gel Electrophoriesis) methods were performed on the materials from patients were results. 1 Vibrio parahaemolyticus didn't grow in 0% density of NaCl, but the fact was found that those grew in 8% density of NaCl. 2. O:K serotypes of Vibrio parahaemolyticus was turned up in domestic patients was 17 types. Among those O3:K6 was the most, it was 68.3%. 3. In 18 kinds of antibiotic tests resistant against Ampicillin, Ticacillin was comparatively high. the case of resistant against Ampicillin, Ticacillin, Vancomycin at the multiple resistant was 52.5%. 4. Toxin gene tdh had only 109 strains among 120 ones isolated from patients held the genes of 199bp size, and 11 strains was negativity 5. In the test of Kanagawa toxic productivity, 107 strains among strains isolated from patients appeared to be positivity reaction 6. The strain that held trh toxin was only 3, and those among test strains had the genes of 250bp size and that had tdh, trh genes at a time were 3 strains, and TDH toxic productivity of those were 16 times, and it was weak. 7. Group Specific-PCR appeared to be useful in the confirmation of O3:K6 serotype interrelations. 8. Three strains which showed difference of 7 DNA sequence even in the same serotype were detected by the result of analyzing the regular gene, toxRS DNA sequence. These strains are differ from general strains which carry infection easily. 9. These mutual dose epidemiological relations were classified into smaller-parts through PFGE method. As a result of such classify, 3 findings were found. V. parahaemolyticus sampled from diarrhea patients were classified into 3 types. And third, the result obtained through PFGE method can be used as a useful tool in a point of molecular-epidemiological view.

The Role of Neutrophils and Epidermal Growth Factor Receptors in Lipopolysaccharide-Induced Mucus Hypersecretion (리포다당질 (lipopolysaccharide)에 의한 기관지 점액 생성 기전에서 호중구와 상피세포 성장인자 수용체 (epidermal growth factor receptor)의 역할)

  • Bak, Sang Myeon;Park, Soo Yeon;Hur, Gyu Young;Lee, Seung Heon;Kim, Je Hyeong;Lee, Sang Yeub;Shin, Chol;Shim, Jae Jeong;In, Kwang Ho;Kang, Kyung Ho;Yoo, Se Hwa
    • Tuberculosis and Respiratory Diseases
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    • v.54 no.1
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    • pp.80-90
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    • 2003
  • Background : Goblet cell hyperplasia is a critical pathological feature in hypersecretory diseases of the airways. A bacterial infection of the lung is also known to induce inflammatory responses, which can lead to the overproduction of mucus. Recently, mucin synthesis in the airways has been reported to be regulated by neutrophilic inflammation-induced epidermal growth factor receptor (EGFR) expression and activation. In addition, it was reported that migration of the activated neutrophils is dependent on the matrix metalloproteinases (MMPs), especially MMP-9. In this study, bacterial lipopolysaccharide (LPS)-induced goblet cell hyperplasia and mucus hypersecretion by EGFR cascade, resulting from the MMPs-dependent neutrophilic inflammation were investigated in the rat airways. Methods : Pathogen-free Sprague-Dawley rats were studied in vivo. Various concentrations of LPS were instilled into the trachea in $300{\mu}{\ell}$ PBS (LPS group). Sterile PBS ($300{\mu}{\ell}$) was instilled into the trachea of the control animals (control group). The airways were examined on different days after instilling LPS. For an examination of the relationship between the LPS-induced goblet cell hyperplasia and MMPs, the animals were pretreated 3 days prior to the LPS instillation and daily thereafter with the matrix metalloproteinase inhibitor (MMPI; 20 mg/Kg/day of CMT-3; Collagenex Pharmaceuticals, USA). The neutrophilic infiltration was quantified as a number in five high power fields (HPF). The alcian blue/periodic acid-Schiff (AB/PAS) stain were performed for the mucus glycoconjugates and the immunohistochemical stains were performed for MUC5AC, EGFR and MMP-9. Their expressions were quantified by an image analysis program and were expressed by the percentage of the total bronchial epithelial area. Results : The instillation of LPS induced AB/PAS and MUC5AC staining in the airway epithelium in a time- and dose-dependent manner. Treatment with the MMPI prevented the LPS-induced goblet cell hyperplasia significantly. The instillation of LPS into the trachea induced also EGFR expression in the airway epithelium. The control airway epithelium contained few leukocytes, but the intratracheal instillation of LPS resulted in a neutrophilic recruitment. A pretreatment with MMPI prevented neutrophilic recruitment, EGFR expression, and goblet cell hyperplasia in the LPS-instilled airway epithelium. Conclusion : Matrix metalloproteinase is involved in LPS-induced mucus hypersecretion, resulting from a neutrophilic inflammation and EGFR cascade. These results suggest a potential therapeutic role of MMPI in the treatment of mucus hypersecretion that were associated with a bacterial infection of the airways.

Antioxidant capacity and Raw 264.7 macrophage anti-inflammatory effect of the Tenebrio Molitor (갈색거저리(Tenebrio Molitor)의 항산화능과 Raw 264.7 대식세포의 항염증 효과)

  • Yu, Jae-Myo;Jang, Jae-Yoon;Kim, Hyeon-Jeong;Cho, Yong-Hun;Kim, Dong-in;Kwon, O-jun;Cho, Yeong-Je;An, Bong-Jeun
    • Food Science and Preservation
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    • v.23 no.6
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    • pp.890-898
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    • 2016
  • The purpose of this paper is to investigate potential anti-inflammatory and anti-oxidant effects of Tenebrio molitor. Macrophage cell response by outside stimulation leads expression of pro-inflammatory cytokines, such as tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), interleukin-6 (IL-6), $interleukin-1{\beta}$ ($IL-1{\beta}$), and trigger expression of genes which are affected by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), resulting in formation of inflammatory factors like nitric oxide (NO) and Prostaglandin $E_2$ (PGE2). Cell viability was determined by MTT assay. In order to investigate anti-inflammatory agents, the inhibitory effects on the production of lipopolysaccharide (LPS)-induced NO in RAW 264.7 cells were examined. T. Molitor significantly decreased the production of NO in a dose-dependent manner, and also reduced the expression of iNOS, a COX-2 protein. As a result, the levels of protein such as $PGE_2$, iNOS, COX-2 and MARKs were significantly reduced compared to non-treated group in T. Molitor water extract (TDW) treated group. Also, antioxidant effect of T. Molitor were investigated using DPPH, ABTS+ and superoxide anion radical scavenging activity tests in cell-free system. Antioxidant activity of T. molitor was found low in the DPPH radical scavenging test while high in the ABTS+ and superoxide anion radical scavenging activity tests. These results show that TDW could be an effective anti-pro-inflammatory and anti-oxidant agent.

Antiproliferation effects of ethanol extract of garlic peels on human cancer cell lines (마늘껍질 70% 에탄올 추출물의 인간 암세포 증식억제 활성)

  • Son, Dae-Yeul
    • Food Science and Preservation
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    • v.24 no.2
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    • pp.289-293
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    • 2017
  • Ethanol extract of garlic peels (GPE) was investigated for its antiproliferative effects on human cancer cell lines. Human lung cancer cell line A549 treated with $500{\mu}g/mL$ GPE resulted in the growth inhibition of A549 by 90%. In stomach cancer cell AGS proliferation inhibition activity, GPE showed 45% and 71% inhibition of AGS growth at $1,000{\mu}g/mL$ and $2,000{\mu}g/mL$, respectively. GPE inhibited the growth of the breast cancer cells MCF-7 effectively at low concentration and showed 78% and 90% inhibitions of MCF-7 growth at $200{\mu}g/mL$ and $500{\mu}g/mL$, respectively. GPE showed very significant antiproliferation effect on liver cancer cell line Hep3B and inhibited Hep3B cell growth by 57% at $100{\mu}g/mL$, and the inhibition's rate increased up to 87% at $500{\mu}g/mL$. Antiproliferation effect of GPE on colorectal cancer cell HT-29 showed 15% reduction of HT-29 cell growth at $200{\mu}g/mL$ and the growth rate was reduced in a dose dependent manner up to $1,000{\mu}g/mL$. These results indicated that GPE had high antiproliferation effects on breast and liver cancer cell lines at low concentrations ($200{\mu}g/mL$), and by higher concentrations over $500{\mu}g/mL$, GPE inhibited the growth of A549 and HT-29. The results of our study suggested the potential use of garlic peels for use as an excellent antiproliferative substance for human cancer cells.

Antiemetic Effect of Dexamethasone in Dogs Sedated with Xylazine (Xylazine hydrochloride로 진정시킨 개에 대한 Dexamethasone의 항구토 효과)

  • Yang Jung-hoon;Kang Han-sem;Bae Jae-sung;Song Chang-hyun;Kim Jung-eun;Jin Hee-kyung;Jang Kwang-ho
    • Journal of Veterinary Clinics
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    • v.22 no.2
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    • pp.94-99
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    • 2005
  • This prospective study aimed to assess the efficacy of dexamethasone to prevent xylazine induced emesis in dogs. The antiemetic effect of graded, single high-dose intravenous dexamethasone against xylazine hydrochloride was studied. Clinically healthy mixed breed dogs that weighed $ 4.64\pm1.25kg$ were used in this study. Food and water were given 3 hours before the experiment. Venous blood specimens were collected from all experimental animals for hema-tological and blood chemical test pre- and post-experiment. Twenty-eight experimental animals were randomly divided into 4 groups; the group treated with 0.2ml/kg of normal saline (Control group), the groups treated with 1mg/kg (D1 group), 2mg/kg (D2 group) and 4 mg/kg of dexamethasone (D4 group). Three doses of the dexamethasone or normal saline was administered intravenousely to each group and after 5 minutes, xylazine (2.2 mg/kg) was administered intramuscularly. The time until onset of the first emetic episode and rate of emesis were investigated. At the same time, the extent of sedation was scored subjectively 5, 15, 30 and 60 minutes after injection of xylazine hydrochloride using Visual Sedation Score. The time until onset of the first emetic episode was $203.25\pm11.35$ sec in Control group, $187.33\pm48.0l$ sec in D1 group and 218.33± 13.58 sec in D2 group. The rate of xylazine induced emesis were $57\%$ in Control group and $43\%$ in D1 and D2 group respectively. On the other hand, any emetic episodes were not observed in 04 group. At extent of sedation score, all experimental animals especially including the animals in D1 group were highly sedated at 15 minutes after administration of xylazine hydrochloride. Hematological and blood chemical values showed normal ranges pre- and post-experiment. We concluded that prior treatment with 4 mg/kg of dexamethasone hardly caused xylazine-induced emesis without disturbing the sedative effect of xylazine in dogs.

Immunoenhancing Effects of Conjugated Linoleic Acid on Chemotactic Activity of Porcine Peripheral Blood Polymorphonuclear Cells (돼지 말초혈액 다형핵 백혈구의 유주성에 있어서 conjugated linoleic acid의 면역증강효과)

  • Kim, Ju-hyang;Chung, Chung-soo;Lee, Chul-young;Yang, Mhan-pyo
    • Journal of Veterinary Clinics
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    • v.20 no.1
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    • pp.1-6
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    • 2003
  • Immunoenhancing effects of conjugated linoleic acid (CLA) isomers (l0t-l2c CLA, 9c-11t CLA, CLA mixture, 9c-11c CLA and 9t-11t CLA) on chemotactic activity of porcine peripheral blood polymorphonuclear cells (PMN) were examined. The chemotactic activity of PMN was evaluated by a modified Boyden chamber assay. CLA isomers at higher concentration of 50 to 200$\mu$M exhibited a low viability of cells by trypan blue exclusion. CLA isomers were used at concentration of 20uM showing no cytotoxic effect and high cell viability. CLA isomers themselves were not active or slight chemotactic for PMN. But culture supernatant from mononuclear cells (MNC) treated with 10t-12c CLA, 9c-11t CLA and CLA mixture except for 9c-11c. CLA and 9t-11t CLA enhanced remarkably chemotactic activity or porcine PMN PMN migration by culture supernatant from MNC treated with CLA mixture was found to be true chemotaxis by checkboard assay. This migration was also induced by porcine recombinant interleukin (rIL)-8. PMN chemotaxis caused both culture supernatant from MNC treated with CLA mixture and porcine rIL-8 was inhibited in a dose-dependent manner by addition of anti-porcine IL-8 polyclonal antibody. Therefore, these results strongly suggested that CLA (10t-12c CLA, 9c-11t CLA and CLA mixture) could stimulate porcine MNC to release and IL-8 like chemotactic activity.

Anti-Inflammatory and Anti-Oxidative Activity of Methanol Extract from Terminalia chebula Retz., Lavandula spica L., and Dalbergia odorifera T. in RAW 264.7 Cells (가자, 라벤더, 강향의 항염증 및 항산화 활성 검색)

  • Chae, In-Gyeong;Yu, Mi-Hee;Kim, Hyuk-Il;Lee, In-Seon
    • Journal of Life Science
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    • v.21 no.4
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    • pp.561-567
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    • 2011
  • This study was performed to evaluate the anti-inflammatory and antioxidant activities of methanol extract from natural products. Cell viability was determined by MTT assay. The production of NO and TNF-${\alpha}$ were measured by Griess assay and enzyme-linked immunosorbent assay (ELISA). In order to effectively screen for anti-inflammatory agents, we first examined the inhibitory effects of 24 natural products on the production of lipopolysaccharide (LPS)-induced nitric oxide (NO) in RAW 264.7 cells. Three extracts of Terminalia chebula Retz., Lavandula spica L., and Dalbergia odorifera T. significantly inhibited NO production. The three extracts significantly decreased production of NO in a dose-dependent manner. Terminalia chebula Retz. decreased TNF-${\alpha}$ production. Antioxidative effects of the three extracts were measured based on polyphenol and flavonoid contents and DPPH radical scavenging activity assay. The three extracts showed high polyphenol contents as well as strong DPPH scavenging activities. In particular, Terminalia chebula Retz. contained the highest polyphenol and flavonoid levels of 616 and $96\;{\mu}g/mg$, respectively, compared to Lavandula spica L. and Dalbergia odorifera T. As DPPH radical scavensing activities, RC50 values of Terminalia chebula Retz. were $2.09\;{\mu}g/ml$.