• Journal of Food Hygiene and Safety
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• v.30 no.3
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• pp.272-280
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• 2015

A simultaneous official method was developed for the determination of phorate and its metabolites (phorate sulfoxide, phorate sulfone, phorate oxon, phorate oxon sulfoxide, phorate oxon sulfone) in livestock samples. The analytes were quantified and confirmed via liquid chromatograph-tandem mass spectrometer (LC-MS/MS) in positive ion mode using multiple reaction monitoring (MRM). Phorate and its metabolites were extracted from beef and milk samples with acidified acetonitrile (containing 1% acetic acid) and partitioned with anhydrous magnesium sulfate. Then, the extract was purified through primary secondary amine (PSA) and C18 dispersive sorbent. Matrix matched calibration curves were linear over the calibration ranges (0.005-0.5 mg/L) for all the analytes into blank extract with $r^2$ > 0.996. For validation purposes, recovery studies were carried out at three different concentration levels (beef 0.004, 0.04 and 0.2 mg/kg; milk 0.008, 0.04 and 0.2 mg/kg, n = 5). The recoveries were within 79.2-113.9% with relative standard deviations (RSDs) less than 19.2% for all analytes. All values were consistent with the criteria ranges requested in the Codex guidelines. The limit of quantification was quite lower than the maximum residue limit (MRL) set by the Ministry of Food and Drug Safety (0.05 mg/kg). The proposed analytical method was accurate, effective and sensitive for phorate and its metabolites determination and it will be used to as an official analytical method in Korea.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.14 no.2
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• pp.47-58
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• 1981

Relationships between the electrical conductivity and the contents of the chloride, sulfate, calcium, magnesium, sodium, potassium and total major inorganic ions, and between each, chemical conservative constituents were calculated with the data which sampled at the lesions of Mulgeum and between Namji and Wondong from March 1974 to April 1980. Semilogarithmic relations were found between the electrical conductivity and the contents of monovalent ions, and logarithmic relations were found between the electrical conductivity and the contents of divalent ions at the both regions. The relational equations between the electrical conductivity $\lambda_{25}$and the contents of the major inorganic ions at Mulgeum are as follows: $log\;Cl(ppm)\;=\;2.37{\cdot}\lambda_{25}(m{\mho}/cm)+0.733{\pm}0.141$, $log\;SO_4(ppm)=1.12{\cdot}log\lambda_{25}(m{\mho}/cm)+2.14{\pm}0.18$, $log\;Ca(ppm)=0.615{\cdot}log\lambda_{25}(m{\mho}/cm)+1.67{\pm}0.12$, $log\;Mg(ppm)=0.756{\cdot}log\lambda_{25}(m{\mho}/cm)+1.27{\pm}0.11$, $log\;Na(ppm)=2.82{\cdot}\lambda_{25}(m{\mho}/cm)+0.551{\pm}0.133$, $log\;K(ppm)=1.33{\cdot}\lambda_{25}(m{\mho}/cm)+0.136{\pm}0.095$, and total inorganic ions $C(ppm)=399{\cdot}\lambda_{25}(m{\mho}/cm)-0.9{\pm}14.6$. The relational equations between the electrical conductivity ($\lambda_{25}$) and the contents of the major inorganic ions at the region between Namji and Wondong a.e as follows: $log\;Cl(ppm)=4.27{\cdot}\lambda_{25}(m{\mho}/cm)+0.380{\pm}0.138$, $log\;SO_4(ppm)=0.915{\cdot}log\lambda_{25}(m{\mho}/cm)+1.95{\pm}0.18$, $log\;Ca(ppm)=0.756{\cdot}log\lambda_{25}(m{\mho}/cm)+1.74{\pm}0.12$, $log\;Mg(ppm)=1.00{\cdot}log\lambda_{25}(m{\mho}/cm)+1.41{\pm}0.10$. $log\;Na(ppm)=2.47{\cdot}\lambda_{25}(m{\mho}/cm)+0.614{\pm}0.065$, $log\;K(ppm)=1.62{\cdot}\lambda_{25}(m{\mho}/cm)+0.030{\pm}0.060$, and total inorganic ions $C(ppm)=323{\cdot}\lambda_{25}(m{\mho}/cm)+11.7{\pm}9.3$. Logarithmic relations were found between each chemical conservative constituents at Mulgeum and the equations are as follows: $log\;Cl(ppm)=0.711{\cdot}log\;SO_4(ppm)+0.488{\pm}0.206$, $log\;Cl(ppm)=0.337{\cdot}log\;Ca(ppm)+0.822{\pm}0.130$, $log\;Cl(ppm)=0.605{\cdot}log\;Mg(ppm)-0.017{\pm}0.154$, $Cl(ppm)=0.676{\cdot}Na(ppm)+2.31{\pm}4.67$, $log\;Cl(ppm)=0.406{\cdot}log\;K(ppm)-0.092{\pm}0.112$, $log\;SO_4(ppm)=0.378{\cdot}log\;Ca(ppm)+0.721{\pm}0.125$, $log\;SO_4(ppm)=0.462{\cdot}log\;Mg(ppm)+0.107{\pm}0.118$, $log\;SO_4(ppm)=0.592{\cdot}log\;Na(ppm)+0.313{\pm}0.191$, $log\;SO_4(ppm)=0.308{\cdot}log\;K(ppm)-0.019{\pm}0.120$, $Ca(ppm)=0.262{\cdot}Mg(ppm)+0.74{\pm}1.71$. $log\;Ca(ppm)=1.10{\cdot}log\;Na(ppm)-0.243{\pm}0.239$, $Ca(ppm)=0.0737{\cdot}K(ppm)+1.26{\pm}0.73$, $log\;Mg(ppm)=0.0950{\cdot}Na(ppm)+0.587{\pm}0.159$, $log\;Mg(ppm)=0.0518{\cdot}K(ppm)+0.111{\pm}0.102$, and $Na(ppm)=0.0771{\cdot}K(ppm)+1.49{\pm}0.59$. Logarithmic relations were found between each chemical conservative constituents except a relationship between the chloride and calcium contents at the region between Namji and Wondong, and the equations are as follows : $log\;Cl(ppm)=0.312{\cdot}log\;SO_4(ppm)+0.907{\pm}0.210$, $log\;Cl(ppm)=0.458{\cdot}log\;Mg(ppm)+0.135{\pm}0.130$, $Cl(ppm)=0.484{\cdot}logNa(ppm)+0.507{\pm}0.081$, $Cl(ppm)=0.0476{\cdot}K(ppm)+1.41{\pm}0.34$, $log\;SO_4(ppm)=0.886{\cdot}log\;Ca(ppm)+0.046{\pm}0.050$, $log\;SO_4(ppm)=0.422{\cdot}log\;Mg(ppm)+0.139{\pm}0.161$, $log\;SO_4(ppm)=0.374{\cdot}log\;Na(ppm)+0.603{\pm}0.140$, $log\;SO_4(ppm)=0.245{\cdot}log\;K(ppm)+0.023{\pm}0.102$, $log\;Ca(ppm)=0.587{\cdot}log\;Mg(ppm)+0.003{\pm}0.088$, $log\;Ca(ppm)=0.892{\cdot}log\;Na(ppm)+0.028{\pm}0.109$, $log\;Ca(ppm)=0.294{\cdot}log\;K(ppm)-0.001{\pm}0.085$, $log\;Mg(ppm)=0.600{\cdot}log\;Na(ppm)+0.674{\pm}0.120$, $log\;Mg(ppm)=0.440{\cdot}log\;K(ppm)+0.038{\pm}0.081$, and $log\;Na(ppm)=0.522{\cdot}log\;K(ppm)-0.260{\pm}0.072$.

• Applied Biological Chemistry
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• v.21 no.3
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• pp.150-184
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• 1978

Nutritional characteristics and physio-chemical properties of mycelial growth and fruitbody formation of oyster mushroom(Pleurotus ostreatus)in synthetic media, the curtural condition for the commerical production in the rice straw and poplar sawdust media, and the changes of the chemical components of the media and mushroom during the cultivation were investigated. The results can be summarized as follows: 1. Among the carbon sources mannitol and sucrose gave rapid mycelial growth and rapid formation of fruit-body with higher yield, while lactose and rhamnose gave no mycelial growth. Also, citric acid, succinic acid, ethyl alcohol and glycerol gave poor fruit-body formation, and acetic acid, formic acid, fumaric acid, n-butyl alcohol, n-propyl alcohol and iso-butyl alcohol inhibited mycelial growth. 2. Among the nitrogen sources peptone gave rapid mycelial growth and rapid formation of fruit-body with higher yield, while D,L-alanine, asparatic acid, glycine and serine gave very poor fruit-body formation, and nitrite nitrogens, L-tryptophan and L-tyrosine inhibited mycelial growth. Inorganic nitrogens and amino acids added to peptone were effective for fruit-body growth, and thus addition of ammonium sulfate, ammonium tartarate, D,L-alanine and L-leucine resulted in about 10% increase fruit-body yield. L-asparic acid about 15%, L-arginine about 20%, L-glutamic acid, and L-lysine about 25%. 3. At C/N ratio of 15.23 fruit-body formation was fast, but the yield decreased, and at C/N ratio of 11.42 fruit-body formation was slow, but the yield increased. Also, at the same C/N ratio the higher the concentration of mannitol and petone, the higher yield was produced. Thus, from the view point of both yield of fruit-body and time required for fruiting the optimum C/N ratio would be 30. 46. 4. Thiamine, potassium dihydrogen phosphate and magnecium sulfate at the concentration of $50{\mu}g%$. 0.2% and 0.02-0.03%, respectively, gave excellent mycelial and fruit-body growth. Among the micronutrients ferrous sulfate, zinc sulfate and manganese sulfate showed synergetic growth promoting effect but lack of manganese resulted in a little reduction in mycelial and fruit-body growth. The optimum concentrati on of each these nutrients was 0.02mg%. 5. Cytosine and indole acetic acid at 0.2-1mg% and 0.01mg%, respectively, increased amount of mycelia, but had no effect on yield of fruit-body. The other purine and pyrimidine bases and plant hormones also had no effect on mycelial and fruit-belly yield. 6. Illumination inhibited mycelial growth, but illumination during the latter part of vegetative growth induced primordia formation. The optimum light intensity and exposure time was 100 to 500 lux and 6-12 hours per day, respectively. Higher intensity of light was injurous, and in darkness only vegetative growth without primordia formation was continued. 7. The optimum temperature for mycelial growth was $25^{\circ}C$ and for fruit-body formation 10 to $15^{\circi}C$. The optimum pH range was from 5.0 to 6.5. The most excellent fry it-body formation were produced from the mycelium grown for 7 to 10 days. The lesser the volume of media, the more rapid the formation of fruit-body; and the lower the yield of fruit-body; and the more the volume of media, the slower the formation of fruit-body, and the higher the yield of fruit-body. The primordia formation was inhibited by $CO_2$. 8. The optimum moisture content for mycelial growth was over 70% in the bottle media of rice straw and poplar sawdust. 10% addition of rice bran to the media exhibited excellent mycelial growth and fruit-body formation, and the addition of calciumcarbonate alone was effective, but the addition of calcium carbonate was ineffective in the presence of rice bran. 9. In the cultivation experiments the total yield of mushroom from the rice straw media was $14.99kg/m^2$, and from the sawdust media $6.52kg/m^2$, 90% of which was produced from the first and second cropping period. The total yield from the rice straw media was about 2.3 times as high as that from the sawdust media. 10. Among the chemical components of the media little change was observed in the content of ash on the dry weight basis, and organic matter content decreased as the cultivation progressed. Moisture content, which was about 79% at the time of spawning, decreased a little during the period of mycelial propagation, after which no change was observed. 11. During the period from spawning to the fourth cropping about 16.7% of the dry matter, about 19.3% of organic matter, and about 40% of nitrogen were lost from the rice straw media; about 7.5% of dry mallet, about 7.6% of organic matter, and about 20% of nitrogen were lost from the sawdust media. For the production of 1kg of mushroom about 232g of organic matter and about 7.0g of nitrogen were consumed from the rice straw media; about 235g of organic matter and about 6.8g of nitrogen were consumed from the sawdust media, 1㎏ of mushroom from either of media contains 82.4 and 82.3g of organic matter and 5.6 and 5.4g of nitrogen, respectively. 12. Total nitrogen content of the two media decreased gradually as the cultivation progressed, and total loss of insoluble nitrogen was greater than that of soluble nitrogen. Content of amino nitrogen continued to increase up to the third cropping time, after which it decreased. 13. In the rice straw media 28.0 and 13.8% of the total pentosan and ${\alpha}$-cellulose, respectively, lost during the whole cultivation period was lost during the period of mycelial growth; in the sawdust media 24.1 and 11.9% of the total pentosan and ${\alpha}$-cellulose, respectively, was lost during the period of mycelial growth. Lignin content in the media began to decrease slightly from the second cropping time, while the content of reduced sugar, trehalose and mannitol continued to increase. C/N ratio of the rice straw media decreased from 33.2 at spawining to 30.0 at ending; that of the sawdust media decreased from 61.3 to 60.0. 14. In both media phosphorus, potassium, manganese and zinc decreased, at magnesium, calcium and copper showed irregular changes, and iron had a tendency to be increased. 15. Enzyme activities are much higher in the rice straw media than in the sawdust media. CMC saccharifying and liquefying activity gradually increased from after mycelial propagation to the second cropping, after which it decreased in both media. Xylanase activity rapidly and greatly increased during the second cropping period rather than the first period. At the start of the third cropping period the activity decreased rapidly in the rice straw media, which was not observed in the sawdust media. Protease activity was highest after mycelial propagation, after which it gradually decreased. The pH of the rice straw media decreased from 6.3 at spawning to 5.0 after fourth cropping; that of the sawdust media decreased from 5.7 to 4.9. 16. The contents of all the components except crude fibre of the mushroom from the rice straw media were higher than those from the sawdust media. Little change was observed in the content of the components of mushroom cropped from the first to the third period, but slight decrease was noticed at the fourth cropping.

• Journal of Food Hygiene and Safety
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• v.34 no.3
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• pp.227-234
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• 2019

An analytical method was developed for the determination of oxytetracycline in agricultural products using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After the samples were extracted with methanol, the extracts were adjusted to pH 4 by formic acid and sodium chloride was added to remove water. Dispersive solid phase extraction (d-SPE) cleanup was carried out using $MgSO_4$ (anhydrous magnesium sulfate), PSA (primary secondary amine), $C_{18}$ (octadecyl) and GCB (graphitized carbon black). The analytes were quantified and confirmed with LC-MS/MS using ESI (electrospray ionization) in positive ion MRM (multiple reaction monitoring) mode. The matrix-matched calibration curves were constructed using six levels ($0.001{\sim}0.25{\mu}g/mL$) and coefficient of determination ($r^2$) was above 0.99. Recovery results at three concentrations (LOQ, $10{\times}LOQ$, and $50{\times}LOQ$, n=5) were from 80.0 to 108.2% with relative standard deviations (RSDs) less than of 11.4%. For inter-laboratory validation, the average recovery was in the range of 83.5~103.2% and the coefficient of variation (CV) was below 14.1%. All results satisfied the criteria ranges requested in the Codex guidelines (CAC/GL 40-1993, 2003) and the Food Safety Evaluation Department guidelines (2016). The proposed analytical method was accurate, effective and sensitive for oxytetracycline determination in agricultural commodities. This study could be useful for safety management of oxytetracycline residues in agricultural products.

• Applied Biological Chemistry
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• v.44 no.3
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• pp.148-152
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• 2001

This study investigated the role of excitatory amino acid systems in the initiation of organophosphate-induced seizures and brain damages in rats through quantitative in vivo microdialysis. Microdialysates were collected from the hippocampus of rat brain, treated with diisopropylfluorophosphate (DFP; 2.67 mg/kg, s.c.) alone, and/or atropine sulfate (15 mg/kg, i.m.) and procyclidine (30 mg/kg, i.m.). The protective effects of atropine, a muscarinic blocker, and/or procyclidine, a N-methyl-D-aspartate and cholinergic antagonist, against DFP were examined. DFP treatment increased the levels of aspartate (Asp) and glutamate (Glu) significantly in the hippocampal persuate with the induction of seizures. Treatment of procyclidine could effectively block the increase of Asp and Glu levels. Atropine treatment showed no significant anticonvulsive effects against DFP-induced seizures. The increases of Asp and Glu levels by DFP were also completely blocked through the combined treatment of atropine and procyclidine. Histopathological findings on the hippocampus confirmed the above results. More effective protection was observed through the treatments of procyclidine alone or of both procyclidine and atropine than atropine alone against DFP-induced brain damage. Procyclidine was shown to be effective in DFP-induced seizures.

• Korean Journal of Agricultural Science
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• v.16 no.2
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• pp.225-238
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• 1989

These studies were conducted to investigate the extraction, purification and characterization of oriental pear (Niitaka. Pyrus pyrifolia Nak.) protease, and the results obtained were as follows: 1. Oriental pear protease was effectively extracted by the method of homogenizing pear pulp with 0.7 volume of 0.1M-sodium phosphate buffer, pH 6.5 containing 5mM-cysteine, 40mM-2-mercaptoethanol and 2mM-EDTA at 10,000 rpm for 5 min. 2. The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography, and the purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. 3. The specific activity of purified enzyme was 29.65 unit/mg protein and the yield was 7.22%. 4. The moecular weight of the protease was estimated to be about 51,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had Km value of 54.5 mg/ml for casein. 5. The purified enzyme had a maximum activity at pH 6.0 and $50^{\circ}C$, and was stable from pH 5.5-6.5 and at temperatures below $50^{\circ}C$ 6. Casein was a better substrate for this protease compared to hemoglobin. 7. The enzyme activity was markedly inhibited by p-chloromercuribenzoic acid and heavy metal salts such as $HgCl_2$ and $MnSO_4$ also considerably inhibited the enzyme activity.

• Korean Journal of Food Science and Technology
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• v.7 no.4
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• pp.229-237
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• 1975

An attempt was made to utilize the enzyme produced by Asp. oryzae as meat tenderizer. The production, purification, and various properties of proteinase produced by Asp. oryzae were investigated. Results obtained are as follow; 1. A strain which had the highest proteolytic activity was selected among 9 Aspergillus species. 2. Culture medium consisted of wheat bran 10g, 2% glucose, 0.03% urea and 0.1% $MgSO_4$ (pH 6.5). Mold was incubated at $30^{\circ}C$ for 3 days. 3. Enzyme extract from culture medium were fractionated with ammonium sulfate and purified by Sephadex G-75 column chromatography. 4. When pH of reaction mixture was controlled, maximal activity of proteinase by Asp. oryzae was obtained at pH 3, pH 6.6, $8.4{\sim}8.5$ and pH 10.0 to 10.5. Those results were interpreted to show that enzyme consists of acid proteinase, neutral proteinase and alkaline proteinase. Enzyme was stable at pH 6 to 10. 5. Opt. temperature for proteinase activity was $50^{\circ}C$, but enzyme was stable up to $40^{\circ}C$. 6. The proteinase was inhibited by $Ag^+$. It was also inhibited by EDTA. 7. When myofibrillar proteins were treated by proteinase from Asp. oryzae, ATPase activities of myofibrillar proteins changed remarkably. Accordingly, it was concluded that proteinase produced by Asp. oryzae were able to be used as meat tenderizer.

• Economic and Environmental Geology
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• v.47 no.4
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• pp.317-333
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• 2014

The Pocheon iron (-copper) deposit, located at the northwestern part of the Precambrian Gyeonggi massif in South Korea, genetically remains controversial. Previous researchers advocated a metamorphosed (-exhalative) sedimentary origin for iron enrichment. In this study, we present strong evidences for skarnification and Fe mineralization, spatially associated with the Myeongseongsan granite. The Pocheon deposit is composed of diverse carbonate rocks such as dolostone and limestone which are partially overprinted by various hydrothermal skarns such as sodic-calcic, calcic and magnesian skarn. Iron (-copper) mineralization occurs mainly in the sodic-calcic skarn zone, locally superimposed by copper mineralization during retrograde stage of skarn. Age data determined on phlogopites from retrograde skarn stage by Ar-Ar and K-Ar methods range from $110.3{\pm}1.0Ma$ to $108.3{\pm}2.8Ma$, showing that skarn iron mineralization in the Pocheon is closely related to the shallow-depth Myeongseongsan granite (ca. 112 Ma). Carbon-oxygen isotopic depletions of carbonates in marbles, diverse skarns, and veins can be explained by decarbonation and interaction with an infiltrating hydrothermal fluids in open system ($XCO_2=0.1$). The results of sulfur isotope analyses indicate that both of sulfide (chalcopyrite-pyrite composite) and anhydrites in skarn have very high sulfur isotope values, suggesting the $^{34}S$ enrichment of the Pocheon sulfide and sulfate sulfur was derived from sulfate in the carbonate protolith. Shear zones with fractures in the Pocheon area channeled the saline, high $fO_2$ hydrothermal fluids, resulting in locally developed intense skarn alteration at temperature range of about $500^{\circ}$ to $400^{\circ}C$.

• Journal of the Mineralogical Society of Korea
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• v.6 no.2
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• pp.122-144
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• 1993

The Kimhae napseok clay deposit was studied to characterize its mineralogy and genesis. Geology of the deposit is composed of Tertiary volcanic rocks and granodiorite. Tertiary volcanic rocks consist of andesitic tuff with minor interstratified tuffaceous shale, and rhyodacitic tuff. The main ore body of 2.4 to 4 m in thickness developed parallel to the bedding of andesitic tuff bed. Its strike and dip are $N70^{\circ}E-N85^{\circ}E$ and $16^{\circ}NW-32^{\circ}NW$, respectively. Two alteration zones; the propylitic zone of albite-epidote-chlorite-quartz assemblage and advanced argillic zone of pyrophyllite-dickite-alunite-diaspore assemblage are developed. Correlation of $SiO_2$ to $Al_2O_3$ shows no relation in propylitic zone, while a negative linear relation in advanced argillic zone. Chemical variation shows that $SiO_2$, $Al_2O_3$, MgO, CaO, $Na_2O$ and $K_2O$ were leached out during hydrothermal alteration. Pyrophyllite, the most abundant mineral in advanced argillic zone, occurs as low temperature 2M polytype. It is closely associated with dickite, diaspore and alunite. The Hinckley index of dickite is 0.83 showing moderate crystallinity. Na content is increasing in the M site with the increasing content of cations in the R-site. the mole percent of Na replacing K in alunite ranges from 53.2 to 71.6. It is also found that pyrophyllite grows in the dissolution site of diaspore. Plagioclase was albitized. Lowering of pH caused mainly by sulfide and sulfate decomposition resulted in preferential leaching of Si. It is inferred that aluminum released from plagioclase in the volcanic rocks as well as from the tuffaceous shale intercalated in andesitic tuff were the main sources of aluminum required for the formation of clay deposit. pH in hydrothermal fluid decreased from propylitic zone to advanced argillic zone with increasing degree of alteration. Based on experimental data reported in the literature and mineral assemblages, the formation temperature of the deposit ranges 270 to $320^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.355-362
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• 2016

The phosphomannomutase/phosphoglucomutase gene (pmm/pgm) of Vibrio anguillarum (the causative agent of fish vibriosis) was cloned, and the open reading frame corresponded to a protein with 446 amino acids. The pmm/pgm gene showed a significant degree of sequence homology with the previously reported genes from V. mimicus, V. vulnificus, V. splendidus, and V. harveyi, with 92.3%, 91.4%, 89.9%, and 89.9% amino acid identity, respectively. By reverse transcriptase-polymerase chain reaction, we found that the pmm/pgm gene was upregulated under cold stress condition. The PMM/PGM protein is known to catalyze the interconversion between mannose-1-phosphate and mannose-6-phosphate or glucose-1-phosphate and glucose-6-phosphate, which are important intermediates for lipopolysaccharide (LPS) biosynthesis. To confirm the role of PMM/PGM in the LPS biosynthetic pathway, we constructed a knock out mutant by homologous recombination. The respective LPSs were isolated from the V. anguillarum wild-type and mutant strains, and changes were compared by subjecting them to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on the different patterns of the LPSs, we expect the pmm/pgm gene to have an important role in LPS biosynthesis. The pmm/pgm-deficient mutant of V. anguillarum will contribute to further studies about the role of LPS in V. anguillarum pathogenesis.