• Title/Summary/Keyword: E. coli system

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Cloning and Expression of Bacillus thuringiensis crylAa1 Type Gene. (Bacillus thuringiensis crylAa1 Type Gene의 클로닝과 발현)

  • 이형환;황성희;권혁한;안준호;김혜연;안성규;박수일
    • Microbiology and Biotechnology Letters
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    • v.32 no.2
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    • pp.110-116
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    • 2004
  • The over-expression in E. coli of the pHLN1-SO(+) and pHLN2-80(-) plasmids cloned an insecticidal crystal protein (ICP) gene (crylAal type) from Bacillus thuringiensis var. kurstaki HD 1 was investigated through in part, the deletion of -80 bp promoter and an alternative change of cloning vector system. Two recombinant plasmids were constructed in an attempt to analyze the over-expression of the ICP in relations to its gene structure possessing only -14 bp [Shine-Dalgarno (SD) sequence of -80 bp promoter]. Also, anther two recombinant plasmids similarly cloned the icp gene in a different vector system. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone, pHLRBS1-14 clone in which only the SD sequence in the inverted orientation icp gene appeared, was more evident than the pHLRBS2-14 clone in which only the -14 bp SD sequence of the right orientated icp gene was shown to exist. The pHLN2-80(-) clone produced more ICP proteins than the pHLRBS1-14 clone. In the two clones, pHLNUC1-80 right-oriented icp gene and the pHLNUC2-80 clone inverted-orientation icp gene in a new different vector, the pHLNUC2-80 produced more ICP proteins in E. coli system. These results indicate that the P/ac promoter, the inverted icp gene insertion and -80 bp promoter (-66 bp part of the icp gene promoters), were concerned with the expression of the icp gene in the recombinant plasmids. In addition, the expression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

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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Antibacterial Activities of Bamboo Sap Against Salmonella Typhimurium and Inhibitory Effects in a Model Food System (죽력의 Salmonella typhimurium 등에 대한 항세균 활성과 Model Food System에서의 생육억제 효과)

  • Chung, Hee-Jong;Ko, Bong-Guk
    • Journal of the Korean Society of Food Culture
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    • v.20 no.6
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    • pp.709-714
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    • 2005
  • Antibacterial activities of the freeze-dried bamboo sap dissolved into the water or 50% ethanol were determined and antimicrobial activity of bamboo sap dissolved into distilled water was most strong with 15 mm of the diameter of inhibiting clear zone against Listeria monocytogenes ATCC 19114 among gram positive bacteria tested, but it did not inhibit Bacillus subtilis ATCC 6633 at all, and the sap was most greatly inhibited the growth of Shigella dysenteriae ATCC 9361 among gram negative bacteria with 15 mm of the diameter of inhibiting clear zone. Bamboo sap dissolved into 50% ethanol most strongly inhibited the growth of L. monocytogenes ATCC 19114 and it also inhibited the growth of B. subtilis ATCC 6633 which did not show any with the sap dissolved into distilled water. The sap dissolved into 50% ethanol was most greatly inhibited the growth of S. dysenteriae ATCC 9361 among gram negative bacteria with 23 mm of the diameter of inhibiting clear zone, and it inhibited Vibrio parahaemolyticus WSDH 22, Vibrio vulnilicus ATCC 29307 and Escherichia coli O157 WSDH 54 with 16 mm of the diameter of inhibiting clear zone. However, Both of the saps dissolved in distilled water and 50% ethanol did not showed any inhibition against the lactic acid bacteria of Lactobacillus plantarum KCTC and Lactobacillus brevis KCTC. Most of the tested bacteria were more sensitive to the sap dissolved in 50% ethanol than the sap dissolved in distilled water. The lowest minimum inhibitory concentration of the bamboo sap dissolved into 50% ethanol was 0.6 mg eq./disc with L. monocytogenes ATCC 19114, but that of the sap dissolved into distilled water was 0.8 mg eq./disc with Staphylococcus epidermides ATCC 12228, S. dysenteriae ATCC 9361, L. monocytogenes ATCC 19114, Salmonella typhimurium WSU 2380 and V. parahaemolyticus WSDH 22. In a model food system of the sterilized chocolate milk, antibacterial activities of the sap dissolved into 50% ethanol were relatively stronger than those of the sap dissolved into distilled water and the activities against the bacteria tested were very similar each other. These result suggested the bamboo sap can be used as a natural food preservative.

HtrA2 Interacts with Aβ Peptide but Does Not Directly Alter Its Production or Degradation

  • Liu, Meng-Lu;Liu, Ming-Jie;Kim, Jin-Man;Kim, Hyeon-Jin;Kim, Jeong-Hak;Hong, Seong-Tshool
    • Molecules and Cells
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    • v.20 no.1
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    • pp.83-89
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    • 2005
  • HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with $A{\beta}$ peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of $A{\beta}$ peptide. The interaction between $A{\beta}$ peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in $A{\beta}$ peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of $A{\beta}$ peptide at the ${\beta}/{\gamma}$-secretase level, or the degradation of $A{\beta}$ peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of $A{\beta}40$ and $A{\beta}42$ by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of $A{\beta}40$ and $A{\beta}42$ suggests that it may play some positive role in mammalian cells.

Proteomic Analysis of Serum Samples from Natural Herb and Immunoglobulin Yolksac (Ig Y) treated Porcine

  • Park, Hyeon-Soo;Nagappan, Arulkumar;Hong, Gyeong-Eun;Yumnam, Silvia;Lee, Ho-Jung;Kim, Gon-Sup
    • Journal of Korean Clinical Health Science
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    • v.1 no.1
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    • pp.83-89
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    • 2013
  • Purpose. Natural herb has been used for traditional medicine for thousands of years. For this reason, alternative medicine methods affect to domestic animal field. The main purpose of this study was to confirm the regulated proteins after feed additive treatment. Methods. We used total thirty male pigs were used for this experiment. E. coli and Salmonella typhimurium, were used for promote enteritis. Animals were divided into a negative control group, positive control group and test group to determine the effect of an additive mixture on the changes in protein expression in serum. The pigs were given a food supplemented with a natural herbal additive containing immunoglobulin yolksac (IgY) at concentrations 1%. On the 1st week and after eight weeks of feeding, the serums were collected from each group. The changes in the serum proteome as a response to the herbal additive were examined using two-dimensional polyacrylamide gel electrophoresis. Results. A total of 14 differentially expressed protein spots were identified by comparing the protein profiles of the control and additive treated porcine serums. Finally, 7 proteins were detected by MALDI-TOF/MS. Moreover, the proteins detected are involved in a cholesterol control factor, inflammation regulator, Conclusion. These results support of the hypothesis that a natural herbal additive containing IgY can affect the immune regulation system and reduce the pathological process by microbial infections.

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Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

  • Park, Jung-Sun;Park, Soo-Young;Cho, Hyun-Il;Sohn, Hyun-Jung;Kim, Tai-Gyu
    • IMMUNE NETWORK
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    • v.11 no.3
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    • pp.182-189
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    • 2011
  • Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Effects of Pre-Converted Nitrite from Red Beet and Ascorbic Acid on Quality Characteristics in Meat Emulsions

  • Choi, Yun-Sang;Kim, Tae-Kyung;Jeon, Ki-Hong;Park, Jong-Dae;Kim, Hyun-Wook;Hwang, Ko-Eun;Kim, Young-Boong
    • Food Science of Animal Resources
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    • v.37 no.2
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    • pp.288-296
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    • 2017
  • We investigated the effects of fermented red beet extract and ascorbic acid on color development in meat emulsions. The pH of meat emulsions containing red beet extract decreased with an increase in the amount of extract added. The redness of the treated meat emulsions was higher than that of the control with no added nitrite or fermented red beet extract (p< 0.05), though the redness of the meat emulsions treated with fermented red beet extract only was lower than in that treated with both fermented red beet extract and ascorbic acid (p<0.05). The highest VBN, TBARS, and total viable count values were observed in the control, and these values in the meat emulsions treated with fermented red beet extract were higher than in that treated with both fermented red beet extract and ascorbic acid (p<0.05). E. coli and coliform bacteria were not found in any of the meat emulsions tested. Treatment T2, containing nitrite and ascorbic acid, had the highest overall acceptability score (p<0.05); however, there was no significant difference between the T2 treatment and the T6 treatment, which contained 10% pre-converted nitrite from red beet extract and 0.05% ascorbic acid (p>0.05). The residual nitrite content of the meat emulsions treated with ascorbic acid was lower than in those treated without ascorbic acid (p<0.05). Thus, the combination of fermented red beet extract and ascorbic acid could be a viable alternative to synthetic nitrite for the stability of color development in meat emulsions.

Role of dietary nucleotides to mitigate post-weaning stress in newly weaned pigs

  • Shin, Taeg Kyun;Wickramasuriya, Samiru Sudharaka;Cho, Hyun Min;Kim, Eunjoo;Kim, Younghwa;Park, Juncheol;Macelline, Shemil Priyan;Heo, Jung Min;Yi, Young-Joo
    • Korean Journal of Agricultural Science
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    • v.44 no.4
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    • pp.477-486
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    • 2017
  • This review provides an overview of dietary nucleotides as an alternative to in-feed antibiotics for weaning pigs. Dietary nucleotides are composed of DNA or RNA molecules and are normally contained in protein-rich feed ingredient, brewer's yeast, yeast extract, and milk. Weaning pigs are suffering from several stresses, such as environmental challenges (i.e. crowding, transportation, and feeding). Such stressors can damage the intestinal epithelium and cause an invasion by Escherichia coli, secondary inflammatory responses, and post weaning diarrhea. To overcome weaning disorder, people often use antibiotics which reduce symptoms and boost growth performance. However, since antibiotics were banned due to concerns of antibiotic resistant bacteria, researchers are studying alternative materials to antibiotics. Dietary nucleotides are one of the alternative materials for replacing antibiotics and can be used in abnormal conditions, such as weaning diarrhea, low digestibility, and disease condition. Nucleotides have substances that have important roles in cell division and cell growth, affecting growth performance, intestinal condition, and immunological effect at the weaning stage. However, nucleotides' composition is very different between sources and this aspect makes it difficult to utilize nucleotides at the weaning stage. Therefore, this review paper focuses on i) the characteristics and functions of dietary nucleotides and ii) the effect of dietary nucleotides on the growth performance and immune system of pigs.

Optical Characterization of Sensory Rhodopsin II Thin Films using a Near-field Scanning Microwave Microscope (근접장 마이크로파 현미경을 이용한 로돕신의 광학적 특성 연구)

  • Yu, Kyung-Son;Kim, Song-Hui;Yoon, Young-Woon;Lee, Kie-Jin;Lee, Jung-Ha;Choi, Ah-Reum;Jung, Kwang-Hwan
    • Journal of the Korean Institute of Electrical and Electronic Material Engineers
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    • v.20 no.1
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    • pp.80-85
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    • 2007
  • We report the electro-optical properties of the sensory rhodopsin II using a near-field scanning microwave microscope(NSMM). Rhodopsin was known as a photoreceptor pigment with a retinal as a chromophore via a protonated Schiff base and consists of seven ${\alpha}-helical$ transmembrane segments. The sensory rhodopsin II, expressing E. coli UT5600 with endogenous retinal biosynthesis system and purified with $Ni^{-2}-NTA$ affinity chromatography in the presence of 0.02 % DM (Dodecyl Maltoside) from Natronomonas pharaonis. We measured the absorption spectra and the transients difference of sensory rhodopsin II from Natronomonas pharaonis using a UV/VIS spectrophotometer with Nd-Yag Laser (532 nm). The absorption spectra of NpSR II showed a typical rhodopsin spectrum with a left shoulder region and the photointermediates spectra of NpSR II-ground state (${\lambda}max=498\;nm$), NpSR II-M state (${\lambda}max=390\;nm$), and NpSR II-O state (${\lambda}max=550\;nm$) during the photocycle. The observed photocycle reaction was confirmed by measuring the microwave reflection coefficient $S_{11}$ at an operating frequency of f=3.93-3.95 GHz and compared with the results of a photocycle of NpSR II.

Inactivation of Microorganisms in Sewage Using a Pilot Plasma Reactor (Pilot 플라즈마 반응기를 이용한 하수 중 미생물의 불활성화)

  • Kim, Dong-Seog;Park, Young-Seek
    • Journal of Environmental Health Sciences
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    • v.39 no.3
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    • pp.289-299
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    • 2013
  • Objectives: For the field application of the dielectric barrier discharge plasma reactor, scale-up of the plasma reactor is needed. This study investigated the possibility of inactivation of microorganisms in sewage using pilot multi-plasma reactor. We also considered the possibility of degradation of total organic carbon (TOC) and nonbiodegradable matter ($UV_{254}$) in sewage. Methods: The pilot plasma reactor consists of plasma reactor with three plasma modules (discharge electrode and quartz dielectric tube), liquid-gas mixer, high voltage transformers, gas supply equipment and a liquid circulation system. In order to determine the operating conditions of the pilot plasma reactor, we performed experiments on the operation parameters such as gas and liquid flow rate and electric discharge voltage. Results: The experimental results showed that optimum operation conditions for the pilot plasma reactor in batch experiments were 1 L/min air flow rate), 4 L/min liquid circulation rate, and 13 kV electric discharge voltage, respectively. The main operation factor of the pilot plasma process was the high voltage. In continuous operation of the air plasma process, residual microorganisms, $UV_{254}$ absorbance and TOC removal rate at optimal condition of 13 kV were $10^{2.24}$ CFU/mL, 56.5% and 8.6%, respectively, while in oxygen plasma process at 10 kV, residual microorganisms, $UV_{254}$ absorbance and TOC removal rate at optimal conditions were $10^{1.0}$ CFU/mL, 73.3% and 24.4%, respectively. Electric power was increased exponentially with the increase in high voltage ($R^2$ = 0.9964). Electric power = $0.0492{\times}\exp^{(0.6027{\times}lectric\;discharge\;voltage)}$ Conclusions: Inactivation of microorganisms in sewage effluent using the pilot plasma process was done. The performance of oxygen plasma process was superior to air plasma process. The power consumption of oxygen plasma process was less than that of air plasma process. However, it was considered that the final evaluation of air and oxygen plasma must be evaluated by considering low power consumption, high process performance, operating costs and facility expenses of an oxygen generator.