Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.
Kim, Yong-Sang;Yun, Suk-Hyun;Jeong, Do-Yeon;Hahn, Kum-Su;Uhm, Tai-Boong
Korean Journal of Microbiology
/
v.46
no.3
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pp.270-277
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2010
In order to manufacture Bacillus cereus-free fermented soybean products, an antimicrobial agentproducing isolate against B. cereus was obtained from 150 traditionally fermented soybean products. The morphological and biochemical tests and the phylogenetic relationship among 16S rRNA gene sequences indicated that the isolate named as the strain SCK 121057 was most closely related to Bacillus licheniformis. The B. licheniformis isolate began to produce the antimicrobial agent after 48 h of incubation. The agent was nonproteinaceous and insensitive to heat, long term storage and protease K. Electron microscopic observation indicated that the agent attacked the membrane of B. cereus, leaving the ghost cell. The isolate inhibited growth of B. subtilis, Lactobacillus brevis and various types of pathogenic strains including Escherichia coli, E. faecalis, Micrococcus luteus, Staphylococcus aureus, Aspergillus flavus, A. ochraceus, and A. parasiticus as well as B. cereus. After coinoculation of B. licheniformis SCK 121057 and B. cereus in the ratio (as the basis of CFU/g sample) of 10 to 1 on the surface of cooked soybeans, cell numbers of B. cereus had been dramatically reduced after 31 days of incubation compared to those of single inoculation of B. cereus.
First. the purification and analysis of alliin in garlic from different origins by alliin-HPLC determination method were studied. Allinase in garlic was inactivated by heating in boiling water followed by extraction of alliin in garlic with 80% methanol. To remove free amino acids and alliin homologs in garlic, garlic extract was separated by cation exchange column which was packed with amberlite CG-120 resin using 40L d-water as eluent. Alliin in garlic extract was crystallized in a mixture of acetone (50$^{\circ}C$):H$_2$O:acetic acid=70:29:1 and then recrystallized in a mixture of acetone (50$^{\circ}C$):H$_2$O:acetic acid=75:24:1. Obtained alliin was identified by melting point. TLC, microscope observation and mass spectrometry. High performance liquid chromatography (HPLC) following pre-column derivatization of cystein derivatives with o-phthaldialdehyde/2-mercaptoethanol has succeessfully been applied to the analysis of various garlics. Each alliic of standard solution and garlic extract was derivatized to isoindole derivative by o-phthaldialdehyde /2-mercaptoethanol and then analyzed by HPLC. Six point calibration was done by using alliin peak area. Lineality was observed at 0 ∼ 1.0mg/ml of alliin concentration. Weighted regression line function was Y=6254X - 256077. By this function, alliin contents in various garlics were 0.34 ∼ 0.73% fresh weight. Second study was designed to evaluate the effects of garlic extracts of various concentrations on the growth of various pathogenes (Eubacterium limonsum, Bacteroides fragilis, Salmonella typhimurium, Salmonella typhi, Shigella sonnei, Kiebsiella pneumoniae, Enterobacter cloacae, Pserdomonas aeruginosa, Escherichia coli). For antimicrobial effects against microorganism, totally minimal inhibition concentrations (MIC) of alliin were from 5,000 to 20,000ppm. MIC of ethanol extract were 1,250 to 10,000ppm.
Journal of the Korean Society of Food Science and Nutrition
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v.23
no.6
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pp.927-932
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1994
A total of 34 Salmonella sp. an d25 Shigella sp. were isolated from 311 patients with diarrhea. The isolation rates of Salmonella sp. ad Shigella sp. were 10.9% and 8%, respectively. The serogroups of 34 Salmonella sp. were in order of group D(50%), group B(38.25), group E(8.8%) and group C 92, 9%0. the serogroups of 25 Shigella sp. were group D(96%) and group B(4%). Seasonal distribution of isolated Salmonella sp. and Shigella sp. were shown the most high at July, 17.65% and 64%, respectively. Age group distribution of isolated Salmonella sp. and Shigella sp. were shown the most high at twenties and thirties (23.5%), and teens(76%), respectively. The Salmonella isolates were resistant in order of prevalence use of streptomycin(SM) (100%), erythromycin (EM) and movobiocin (NB)(90.6%), penicillin G(PG) (65.6%) and cephalexin (CPX)(46.9%). the isolates of Shigella sp. were resistant in order of prevalence use of EM (95.8%), NB(91.7%), SM(87.5%). Eighteen kinds of resistant patterns of Salmonella ioslates were detected. The multiple resistance patterns of Shigella isolates were mostly SM, EM, NB type (79.2%). The minimum inhibitory concentration of Salmonella sp. and Shigella sp. and Shigella sp. isolated from patients with diarrhea were tabulated.
Kim, Soo Young;Oh, Chang Geun;Lee, Young Joo;Choi, Kyu Ha;Shin, Doo Sik;Lee, Si Kyung;Park, Kab Joo;Shin, Hakdong;Park, Myeong Soo;Lee, Ju-Hoon
Journal of Microbiology and Biotechnology
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v.23
no.4
/
pp.545-554
/
2013
Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.
Journal of the Korean Institute of Telematics and Electronics D
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v.36D
no.8
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pp.36-44
/
1999
This paper presents the design and fabrication of the duplexer for a Ka-band satellite transponder which is analyzed transmission characteristics by calculating the generalized scattering matrix using mode matching method. It is composed of 2 bandpass filters, coupled with H-plane T-junction having symmetrical inductive iris and E-plane metal insert structures. Compared with the size and weight of the Rx and Tx filter loaded with a transponders respectively, those of the duplexer can be effectively reduced. In a high power transmission, the variation of the filter characteristics is minimized by the scheme that irises are extended to the exterior of H-plane of the waveguide. This scheme needs no extra heat sinks for dissipating high power. The duplexer is designed to improve the simplification, durability and reliability by eliminating tuning screws, which have been used to compensate for the characteristics of fabricated filters. The bandpass filters of the duplexer show the insertion loss of less than 1.2 dB and the return loss in excess of 15 dB. The isolations of more than 65 dB have been achieved between Rx and Tx filter.
JSTS:Journal of Semiconductor Technology and Science
/
v.5
no.3
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pp.149-158
/
2005
This paper describes design, fabrication, and application of the silicon based temperature controllable micro reactor. In order to achieve fast temperature variation and low energy consumption, reaction chamber of the micro reactor was thermally isolated by etching the highly conductive silicon around the reaction chamber. Compared with the model not having thermally isolated structure, the thermally isolated micro reactor showed enhanced thermal performances such as fast temperature variation and low energy consumption. The performance enhancements of the micro reactor due to etched holes were verified by thermal experiment and numerical analysis. Regarding to 42 percents reduction of the thermal mass achieved by the etched holes, approximately 4 times faster thermal variation and 5 times smaller energy consumption were acquired. The total size of the fabricated micro reactor was $37{\times}30{\times}1mm^{3}$. Microchannel and reaction chamber were formed on the silicon substrate. The openings of channel and chamber were covered by the glass substrate. The Pt electrodes for heater and sensor are fabricated on the backside of silicon substrate below the reaction chamber. The dimension of channel cross section was $200{\times}100{\mu}m^{2}$. The volume of reaction chamber was $4{\mu}l$. The temperature of the micro reactor was controlled and measured simultaneously with NI DAQ PCI-MIO-16E-l board and LabVIEW program. Finally, the fabricated micro reactor and the temperature control system were applied to the thermal denaturation and the trypsin digestion of protein. BSA(bovine serum albumin) was chosen for the test sample. It was successfully shown that BSA was successfully denatured at $75^{\circ}C$ for 1 min and digested by trypsin at $37^{\circ}C$ for 10 min.
Salmonella infections cause the disease in pigs but also some zoonotic Salmonella serotypes can be transmitted to human through swine products, resulting in food poisoning. The objective of this study was to investigate the bacteriological prevalence and detection of invA gene using Salmonella specific polymerase chain reaction (PCR), the epidemiological characteristics related to Salmonella strains cultured from pig samples in Gangwon areas using serotyping, random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) methods. During the period of November 2001 through April 2002, 1,174 ileocecal lymph node were collected from the slaughtered pigs raised in 38 farms located in Gangwon province. The samples were submerged in boiling water and macerated in saline and lymph node homogenates were inoculated into Tetrathionate broth with iodine (TTB, Difco, 0.5% iodine was added) for enrichment growth. Then additional tests were performed using several mediums, and suspects were identified by API 20E kit (BioMerieux) and PCR. Of total 1,174 samples from 38 farms, 44 (3.7%) were isolated as Salmonella spp from 13 farms (34.2%). Of 44 isolates, 31 were in Yangyang region, followed by 9 in Goseong, 2 in both Gangneung and Sokcho. However, there was no difference in regional isolation frequency. All isolates have a 521bp amplified product in Salmonella specific PCR with primer invA which encodes in proteins for invasion of epithelial cells. Of 44 recovered serotypes, 23 (52.3%) were S Eingedi, 10 (22.7%) S Schwarzengrund, 9 (20.5%) S Typhimurium, and 2 (4.5%) S Mbandaka. In RAPD analysis, there appeared to be unique bands distinguishing each serotype, although similarities exist between the different serotypes. Four serotypes of 44 Salmonella isolates appeared to fall into 14 different RAPD types. In PFGE analysis, 9 S Typhimurium were tested with XbaI enzyme and SpeI enzyme. The combination of results obtained with two enzymes subdivided the 9 S Typhimurium into 4 PFGE types.
A study was undertaken to investigate the distribution of Streptomyces spp. in soil in Korea. Among the different types of soil surveyed, the highest population of Streptomyces spp. recording $5.6{\times}10^5\;CFU/g$ was observed in upland soil. With reference to the soil depth, most of their population was distributed from soil surface to 5cm depth and the highest value was found in $0{\sim}2cm$ soil depth. Comparing the population of Streptomyces spp. with different soil color (by Munsell soil color chart), the highest value of $9.2{\times}10^\;CFU/g$ was showed in Oliver yellow soil (2.5Y, 6/4). On the basis of the acidity of soil samples subjected to Streptomyces spp. isolation, it is considered that the optimum pH range for Streptomyces spp. in soil lies between 6.0 to 7.5, showing the highest value of $1.05{\times}10^6CFU/g$ at pH 7.5. Among the colors of isolated colonies, gray and white colonies occupied 60% and 26% of the total isolates respectively.
As a material of metal-ceramic prosthesis, nickel as a form of Ni-Cr alloy has been used for many dental prostheses in many cases. However, several problems in use of the alloy have been revealed (ex ; tissue stimulation, skin allergy, hypersensitivity cytotoxicity and carcinogenecity). Little is known about nickel with respect to the relationship between Ni-prosthesis and gaining of Niresistance in oral microorganisms. The present study was undertaken to check whether use of Ni-prosthesis leads to occurrence of Ni-resistant microorganisms. So this study may suggest the possible relationships between the oral microorganisms and nickel-resistance in oral environment. Bacteria were isolated from the gingival crevicular fluid on the patients wearing Ni-Cr prosthesis. The isolated bacteria were tested fir their Ni-resistance in nickel containing media at different concentration from 3mM to 110mM. E. coli HB101 was used as control. The Ni-resistant bacteria were isolated and biochemically identified. The Ni-resistant bacteria were tested several biochemical, molecular-biological tests. Performed tests were : measuring the growth curve, antibiotic test, growth ability test in liquid media, isolation of the chromosome and plasmid, digestion of DNA by restriction enzyme, electrophoresis of chromosome and plasmid DNA, identification of Ni-resistant genes by the DNA hybridization. The results were as follows 1) The bacteria isolated from gingival crevicular fluid on the patients wearing Ni-Cr alloy prosthesis showed nickel-resistance. 2) The isolated microorganisms grew at nickel containing media of high concentrations (60mM-110mM). 3) Based on the biochemical tests, the isolated microorganisms were identified as Enterococcus faecalis(13 cases), Klebsiella pneumoniae(1 case) and Enterobacter gergoviae(1 case). 4) Enterococcus faecalis expressed not only nickel resistance but also the multi-drug resistance to several antibiotics ; chloramphenicol, kanamicin, streptomycin, lincomycin, clindamycin, However, all strain showed the sensitivity against the tetracycline. 5) DNA hybridization result suggests that there is no homology between the previously known gene of nickel resistance in Klebsiella pneumoniae and chromosomal DNA of Enterococcus faecalis.
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