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1,25-dihydroxyvitamin D3 affects thapsigargin-induced endoplasmic reticulum stress in 3T3-L1 adipocytes

  • Dain Wi;Chan Yoon Park
    • Nutrition Research and Practice
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    • v.18 no.1
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    • pp.1-18
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    • 2024
  • BACKGROUND/OBJECTIVES: Endoplasmic reticulum (ER) stress in adipose tissue causes an inflammatory response and leads to metabolic diseases. However, the association between vitamin D and adipose ER stress remains poorly understood. In this study, we investigated whether 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alleviates ER stress in adipocytes. MATERIALS/METHODS: 3T3-L1 cells were treated with different concentrations (i.e., 10-100 nM) of 1,25(OH)2D3 after or during differentiation (i.e., on day 0-7, 3-7, or 7). They were then incubated with thapsigargin (TG, 500 nM) for an additional 24 h to induce ER stress. Next, we measured the mRNA and protein levels of genes involved in unfold protein response (UPR) and adipogenesis using real-time polymerase chain reaction and western blotting and quantified the secreted protein levels of pro-inflammatory cytokines. Finally, the mRNA levels of UPR pathway genes were measured in adipocytes transfected with siRNA-targeting Vdr. RESULTS: Treatment with 1,25(OH)2D3 during various stages of adipocyte differentiation significantly inhibited ER stress induced by TG. In fully differentiated 3T3-L1 adipocytes, 1,25(OH)2D3 treatment suppressed mRNA levels of Ddit3, sXbp1, and Atf4 and decreased the secretion of monocyte chemoattractant protein-1, interleukin-6, and tumor necrosis factor-α. However, downregulation of the mRNA levels of Ddit3, sXbp1, and Atf4 following 1,25(OH)2D3 administration was not observed in Vdr-knockdown adipocytes. In addition, exposure of 3T3-L1 preadipocytes to 1,25(OH)2D3 inhibited transcription of Ddit3, sXbp1, Atf4, Bip, and Atf6 and reduced the p-alpha subunit of translation initiation factor 2 (eIF2α)/eIF2α and p-protein kinase RNA-like ER kinase (PERK)/PERK protein ratios. Furthermore, 1,25(OH)2D3 treatment before adipocyte differentiation reduced adipogenesis and the mRNA levels of adipogenic genes. CONCLUSIONS: Our data suggest that 1,25(OH)2D3 prevents TG-induced ER stress and inflammatory responses in mature adipocytes by downregulating UPR signaling via binding with Vdr. In addition, the inhibition of adipogenesis by vitamin D may contribute to the reduction of ER stress in adipocytes.

Changes of the blood chemistry, lipid and protein components in blood and liver tissue of the rat after oral combined administration of caffeine, iron and vitamin E (Caffeine, 철분 및 vitamin E 혼합투여시 rat의 혈액과 간조직내에서 혈액화학성분과 지질 및 단백질 구성성분의 변화)

  • Do, Jae-cheul;Huh, Rhin-sou
    • Korean Journal of Veterinary Research
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    • v.36 no.3
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    • pp.577-598
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    • 1996
  • This study was conducted to identify the effects of caffeine or combinations of caffeine and iron or vitamin E on the lipid and protein components or blood chemistry levels of the serum as well as the total homogenate, mitochondrial and microsomal fraction of the rat(Sprague-Dawley, female) liver. Chronic test were conducted to determine those effects. The chronic test was conducted by dividing rats into 5 groups according to the type of drugs and dosages administrated as follows; the control(group A), and group B was given 25mg/kg caffeine orally once daily for 30 days, group C was given 50mg/kg caffeine orally once daily for 30 days, group D was given 25mg/kg caffeine and orally ferric chloride once daily for 30 days and group E was given 25mg/kg caffeine and 25mg/kg vitamin E once daily for 30 days. The concentrations of glucose, urea nitrogen, uric acid, creatinine, total protein, albumin, A/G ratio, triglyceride, total cholesterol, HDL-cholesterol, free fatty acid, phospholipid as well as the activities of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP) were measured in the serum of each experimental groups. The concentrations of the carbonyl group and malondiaidehyde(MDA) and the patterns of the SDS-PAGE(Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) and fatty acid compositions in free fatty acids and phospholipids were analyzed to determine the oxidative damages and metabolic changes on the lipid and protein components in the serum, and total homogenate, mitochondrial and microsomal fractions of the rat liver. The results obtained from this study were summarized as follows; 1. Body weights of groups B, C, D and E were significantly decreased(p < 0.01) in comparison with that of the control in the chronic test. 2. The concentrations of serum glucose in groups B(124.5mg/dl), C(130.1mg/dl), D(122.1mg/dl), E(119.3mg/dl) were significantly higher(p < 0.01) in comparison to that of the control(101.5mg/dl). But, there were no significant differences in the concentrations of urea nitrogen, uric acid, creatinine, total protein, albumin and A/G ratio in comparison to that of the control. 3. The concentrations of total cholesterol and HDL-cholesterol in serum of groups B(69.6, 53.4mg/dl), C(73.0, 56.3mg/dl), D(68.9, 51.1mg/dl) and E(68.2, 51.3mg/dl) were significantly higher(p < 0.01) in comparison to that of the control(52.6, 38.8mg/dl). On the other hand, the concentrations of triglyceride in serum of groups B(45.0mg/dl), C(40.4mg/dl), D(33.8mg/dl) and E(47.2mg/dl) were significantly lower(p < 0.01) in comparison to that of the control(66.2mg/dl). There were no significant differences in the activities of ALT, AST and ALP in comparison to that of the control. 4. The concentrations of free fatty acid and phospholipid in serum of groups B(45.7, 154.4mg/dl), C(50.0, 167.2mg/dl), D(52.5, 148.4mg/dl) and E(41.1, 159.2mg/dl) were higher(p < 0.01) in comparison to that of the control(35.2, 125.3mg/dl). And the concentrations of the carbonyl group and malondialdehyde in serum of group D(1.82, 0.52nM/mg protein) were significantly higher(p < 0.01) in comparison to the control(1.53nM/mg protein). 5. The concentrations of carbonyl group in total homogenate, mitochondrial and microsomal fraction of group D(1.45, 0.94, 1.67nM/mg protein) were significantly higher (p < 0.01) in comparison to the control(1.16, 0.66, 1.27nM/mg protein). And the concentrations of malondialdehyde in the total homogenate, mitochondrial and microsomal fraction of group D(6.70, 6.10, 1.36nM/mg protein) were significantly higher(p < 0.01) in comparison to the control(5.17, 3.64, 0.68nM/mg protein). 6. As the analytical results of the fatty acid compositions of free fatty acid in serum, the proportions of stearic acid and arachidonic acid of groups B(16.52, 12.62%), C(17.52, 15.18%), D(19.73, 13.47%) and E(17.62, 13.28%) were significantly higher(p < 0.01) in comparison to the control(14.75, 7.88%), but the proportions of oleic acid and linoleic acid of groups B(12.97, 32.59%), C(10.88, 31.23%), D(12.37, 30.66%) and E(11.95, 32.41%) were significantly lower(p < 0.01) in comparison to the control(16.44, 35.12%). Otherwise, as the results of the fatty acid compositions of phospholipid in serum, the proportions of stearic acid and arachidonic acid of groups B(39.37, 16.39%), C(40.63, 17.83%), D(42.73, 15.39%) and E(39.16, 15.70%) were significantly higher(p < 0.01) in comparison to the control(37.74, 14.24%), but the proportions of oleic acid and linoleic acid of groups B(4.03, 14.38%), C(3.54, 12.38%), D(4.52, 11.68%) and E(4.29, 13.64%) were significantly lower(p < 0.01) in comparison to the control(5.53, 16.14%). 7. As the analytical results of the fatty acid compositions of free fatty acid in total homogenate, mitochondrial and microsomal fraction of liver, the proportions of oleic acid of groups B(7.8**, 8.73**, 6.88%) and C(6.89**, 7.75**, 6.58%) were lower(**:p < 0.01) in comparison to the control(8.67, 10.08, 7.81%), but the proportions of arachidonic acid of group C(22.62, 19.79, 23.71%) were significantly higher(p < 0.01) in comparison to the control(20.93, 18.47, 22.24%). And the proportions of palmitic acid of group D(25.95**, 26.16, 26.34**%) were significantly higher(**:p < 0.01) in comparison to the control(24.43, 25.42, 23.34%). In addition, the proportions of linoleic acid of group D(23.43, 25.02, 23.95%) were also significantly higher(p < 0.01) in comparison to the control(22.17, 23.75, 21.26%). The proportions of stearic acid of group D(19.87, 19.76**%) in mitochondrial and microsomal fraction were lower(**:p < 0.01) in comparison to the control(21.01, 24.18%), and the proportions of stearic acid of group E(16.71*, 19.65**%) in mitochondrial and microsomal fraction were significantly lower(**:p < 0.01, *:p < 0.05) in comparison to the control(21.01, 24.18%), and the proportions of linoleic acid of group E(25.04, 29.20, 26.48%) in total homogenate, mitochondria and microsome were significantly higher(p < 0.01) in comparison to the control(22.17, 23.75, 21.26%). 8. As the results of the fatty acid compositions of phospholipid in total homogenate, mitochondrial and microsomal fraction of liver, the proportions of palmitic acid of group D(17.58**, 18.78*, 18.23%**) were significantly higher(**:p < 0.01, *:p < 0.05) in comparison to the control(16.28, 17.22, 16.38%), and the proportions of stearic acid of group D(36.41, 37.23, 39.53%) were also significantly higher(p < 0.01) in comparison to the control(34.18, 34.16, 36.04%). But the proportions of oleic acid(3.41*, 3.11**, 3.12**%) and linoleic acid (18.03**, 15.79**, 14.74**%) of group D were significantly lower(**:p < 0.01, *:p < 0.05) in comparison to the control(oleic : 3.63, 3.72, 3.79%, linoleic : 20.03, 18.71, 18.48%). 9. In order to determine the oxidative damages to the protein in serum, mitochondrial and microsomal fraction of the rat liver, the patterns of the SDS-PAGE were identified, but the results of SDS-PAGE were not significantly different between the control and experimental groups.

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Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis

  • Kim, Se-Yu;Park, Soo-Young;Choi, Soo-Keun;Park, Seung-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.25 no.7
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    • pp.1015-1025
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    • 2015
  • The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

Electronic Structures of Co-Pd Alloy Films Using Synchrotron Radiation Photoemission Spectroscopy (방사광 광전자 분광법을 이용한 Co-Pd 합금박막의 전자구조 연구)

  • 강정수;권세균;하양장;민병일;조용필;이창섭;정인범;구양모;김건호
    • Journal of the Korean Magnetics Society
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    • v.6 no.6
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    • pp.405-410
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    • 1996
  • Valence band photoemission spectroscopy (PES) measurements have been performed for $Co_{x}Pd_{100-x}$ alloy films using synchrotron radiation (x = 0, 25, 40, 65). Then the partial spectral weight distributions (PSW's) of Co 3d and Pd 4d electrons have been determined. The Co 3d PSW's exhibit some structures which are quite different from those of the Co film for x < 25 %, whereas they become very similar to those of the Co film for x > 40 %. For x < 25 %, the peak near the Fermi level ($E_F$) and a shoulder around 2 eV binding energy in the Co 3d PSW reflect large hybridization between Pd 4d and Co 3d electrons, suggesting that the hybridization might play an inportant role in determining perpendicualr magnetic anisotropy. The Pd 4d PSW's in Co-Pd alloy films are found to have larger FWHM's (full widths at half maximum), larger binding energies of the main peaks, and larger spectral intensities at $E_F$ than the PES spectrum of the Pd film. The FWHM of the Pd 4d PSW increases with decreasing Pd concentration, which are considered to reflect the disordering effect in the alloy formation or the change in the Pd 4d electronic structure due to hybridization between Co 3d and Pd 4d electrons.

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The Effects of 1,25- Dihydroxyvitamin $D_3$ on Expression of IGF-I Gene and Cellular Proliferation in MC3T3-E1 Cells (골아세포의 IGF-I 유전자 발현 및 세포증식에 대한 1,25-dihydroxyvitamin $D_3$의 영향)

  • Choi, Hee-Dong;Lee, Jae-Mok;Suh, Jo-Young
    • Journal of Periodontal and Implant Science
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    • v.30 no.1
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    • pp.39-52
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    • 2000
  • Polypeptide growth factor belong to a class of potent biologic mediator which regulate cell differentiation, proliferation, migration and metabolism. 1,25-dihydroxyvitamin $D_3$ decrease cell proliferation, and stimulate alkaline phosphatase activity which express in osteoblast during cell differentiation period. IGF-I is known to stimulate cell proliferation and differentiation too. 1,25-dihydroxyvitamin $D_3$ is known to increase IGF-I binding sites and IGF binding protein which inhibite the effect of IGF. The purpose of this study is to evaluate potential role of IGF-I as mediator that control the action of 1,25-dihydroxyvitamin $D_3$. MC3T3-E1 cell were seeded $5{\times}10^5/ml$ at 100mm culture plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 5% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ added. Total mRNA was extracted at 0, 6, 24, 48, 72 hour. PRPCR method was programed for the detection of IGF-I mRNA. In the both groups of 1,25-dihydroxy vitamin $D_3$ treated and control, alternative splicing form of IGF-I, IGF-IA and IGF-IB were expressed. In the 1,25-dihydroxyvitamin $D_3$ treated group, IGF-I mRNA expression was matained until 24 hour, there after expression was decresed. MC3T3-E1 cell were seeded $2.5{\times}10^4/ml$ at 24well plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 3% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ and 10 ng/ml IGF-I were added separately or together. Cell were cultured for 1 and 3 days, $2{\mu}Ci/ml\;[^3H]$ -thymidine was added for the last 24h of culture of each days. ${[^3H]}$-thymidine incorporation in to DNA was measured and expressed counter per minute(CPM). DNA synthetic activity was significantly decreased by 1,25-dihydroxyvitamin $D_3$ both at 1 day and 3 day, and in the combination group of 1,25-dihydroxyvitamin $D_3$ and IGF-I, DNA synthetic activity was also decreased both at 1 day and 3 days. IGF-I did not affect the DNA synthetic activity compared to control group both at 1 day and 3 day. From the above results, 1,25-dihydroxyvitamin $D_3$ was potent inhibitor of cell proliferaton in MC3T3-E1 cells. It assumed that the effect of 1,25-dihydroxyvitamin $D_3$ on osteoblast proliferation may be mediated in part by decreased level of IGF-I.

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Trend Analysis of Men's Gymnastics on Floor Exercise in the 2018 Asian Games through Digital Video Materials (디지털 영상자료를 통한 2018 아시안게임 기계체조 마루운동에 대한 연기내용 분석)

  • Han, Yoon-Soo
    • Journal of Digital Convergence
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    • v.16 no.12
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    • pp.619-624
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    • 2018
  • The purpose of this study was to examine the trend of men's gymnastics on floor exercise final(8 gymnasts) in the 2018 Asian Games through digital video materials. Video recordings of floor exercise final were collected from the 2018 Asian Games. The cross-check with three international gymnastics judges on floor exercise final was analyzed. To fulfil the D-score, 8 gymnasts were the most performed backward saltos elements group(36 elements), forward salto with 1/1 turn(8 gymnasts), backward salto with 5/2 turn(8 gymnasts) and backward salto with 3/1 turn(8 gymnasts). The scores showd average the D score 6.0, E score 7.9, and connection values 0.25. To get a good results on floor exercise, gymnasts need to get a average the D score over 6.0, E score over 8.5, and connection values over 0.3.

PROPAGATION OF THE BLUE CRAB, PORTUNUS TRITUBERCULATUS (MIERS) (꽃게 Portunus trituberculatus (MIERS)의 종묘 생산에 관한 연구)

  • PYEN Choong-Kyu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.3 no.3
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    • pp.187-198
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    • 1970
  • The author succeeded in rearing the young blue crab from the first stage of zoe ato the true crab shape, and during this time he observed their growth and metamorphosis. The relationships between the number of eggs carried by female crabs (E) and the carapace width (C) and body weight (W) are shown as follows: E= 27.9049C-281.8155, E=0.5682 W-116.4606. There are five zoeal stages and a megalopa in the complete larval development of the blue crab. Water temperature in rearing aquaria ranged from 21.4 to $25.2^{\circ}C$. The duration of each zoeal stage was two days on the average. After the fifth moulting, the zoea becomes megalopa and 5 to 6 days later the megalopa moults and develops into the first stage of adult crab shape. The carapace width of megalopa measured about 1.70 mm and the carapace length, from the tip of the rostrum to the posterior dorsal margin of the carapace, was about 2.78 mm on the average. The carapace width and length of the first crab, 18 days after hatching, measured about 4.48 mm and 2.62 mm respectively. After two days, the first crab moulted and grew into the second crab with about 6.47 mm in carapace width and 4.66 mm in carapace length. The larval rearing in the outdoor tank shelved better results than in the indoor aquarium. The highest mortality occurred when the first stage of zoea moulted into the second stage. Percentage of crabs which survived, from the first crab to the ninth crab stages, was about $55\%$. The relationships between rearing days (D) and the carapace width (C), carapace length (L) and body weight (W) of the crab stages during 40 days of rearing are shown as follows. Carapace width, Indoor: C=1.1250D+1.7227 Outdoor C=1.3465D -0.2449 Carapace length, Indoor: L=0.6654D+1.6712 Outdoor: L=0.7893D+0.6919 Body Weight, Outdoor: $$W=1.15e^{0.12423D}$$ Indoor: $$W=6.759\times10^{-2}D^{1.2598}$$ (9-19 day old crabs) Outdoor: $$W=4.136\times10^{-2}D^{1.6024}$$ (21-40 day old crabs) During the crab stage, the following relationships between the number of moulting times and the carapace width (C), carapace length (L) and body weight (W) were found as follows: $$C=5.2e^{0.28119N}$$ $$L=3.65e^{0.26372N}$$ $$W= 0.14e^{0.7037N}$$ The relationships between the carapace length (L) and the carapace width (C) and body weight (W) of the crab stages are shown as follows: Carapace length, mm Formula 2.62-27.17 L=1.6864C-1.0387 7.47-18.53 $$W=9.367\times10^{-5}C^{3.5567}$$ 22.11-27.17 $$W=3.406\times10^{-5}C{3.8571}$$

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A New 5α, 8α-Epidioxy Sterol from the Marine Sponge Plakortis simplex

  • Oh, Jung-Soon;Kim, Myoung-Ha;Song, Ah-Rin;Rho, Jung-Rae
    • Journal of the Korean Magnetic Resonance Society
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    • v.14 no.1
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    • pp.1-8
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    • 2010
  • Four 5,8-epidioxy sterols were isolated from the marine sponge Plakortis simplex. Their structures were completely determined by an extensive NMR analysis and comparison with NMR data of similar compounds for absolute stereochemistry of the side chain. The compounds were assigned as 5,8-epidioxy- (24S)-ethylcholesta-6,22(E),25-trien-3-ol(1), 5,8-epidioxy-(24S)-methylcholesta- 6,22(E)-dien-3-ol(2), 5,8-epidioxycholesta-6,22(E)-dien-3-ol(3) and 5,8- epidioxycholesta-6-en-3-ol (4).

Chemical Constituents from Solenostemma argel and their Cholinesterase Inhibitory Activity

  • Demmak, Rym Gouta;Bordage, Simon;Bensegueni, Abederrahmane;Boutaghane, Naima;Hennebelle, Thierry;Mokrani, El Hassen;Sahpaz, Sevser
    • Natural Product Sciences
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    • v.25 no.2
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    • pp.115-121
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    • 2019
  • Alzheimer's disease is a chronic neurodegenerative disorder with no curative treatment. The commercially available drugs, which target acetylcholinesterase, are not satisfactory. The aim of this study was to investigate the cholinesterase inhibitory activity of Solenostemma argel aerial part. Eight compounds were isolated and identified by NMR: kaempferol-3-O-glucopyranoside (1), kaempferol (2), kaempferol-3-glucopyranosyl($1{\rightarrow}6$)rhamnopyranose (3) p-hydroxybenzoic acid (4), dehydrovomifoliol (5), 14,15-dihydroxypregn-4-ene-3,20-dione (6), 14,15-dihydroxy-pregn-4-ene-3,20-dione-$15{\beta}$-D-glucopyranoside (7) and solargin I (8). Two of them (compounds 2 and 3) could inhibit over 50 % of butyrylcholinesterase activity at $100{\mu}M$. Compound (2) displayed the highest inhibitory effect against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with a slight selectivity towards the latter. Molecular docking studies supported the in vitro results and revealed that (2) had made several hydrogen and ${\pi}-{\pi}$ stacking interactions which could explain the compound potency to inhibit AChE and BChE.

Prevalence and Thermal Stability of Enterobacter sakazakii from Unprocessed Ready-to-Eat Agricultural Products and Powdered Infant Formulas

  • Jung, Mi-Kyoung;Park, Jong-Hyun
    • Food Science and Biotechnology
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    • v.15 no.1
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    • pp.152-157
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    • 2006
  • Enterobacter sakazakii, designated as an unique microbial species in 1980, may cause bacteremia, necrotizing enterocolitis and infant meningitis. The distribution and the thermostability of E. sakazakii in unprocessed ready-to-eat (RTE) agricultural products of 252 and in 25 powdered infant formulas (PIF) were analyzed. Eighty one, 50, 43, and 47% of brown rice, pumpkin, potato, and carrot samples, respectively, had aerobic plate counts (ARC) in the range of 5 log CFU/g or more. Almost all the other products sampled had APC of approximately 2 log CFU/g. Fifty three, 75, 67, and 68% of banana, pumpkin, soybean, and carrot had Enterobacteriaceae counts approximating 3 log CFU/g. Sixty six percent of the brown rice tested had Enterobacteriaceae counts approximating 5-6 log CFU/g. E. sakazakii was isolated from 3/25(12%), 4/23(17%), 1/24(4%), and 1/27(4%) of PIF, brown rice, laver, and tomato samples, respectively. D-values were 3.52-4.79 min at 60 and $D_{60}-values$ were similar as the isolates reported. Thermal inactivation of four thermovariant E. sakazakii strains during the rehydration of PIF with hot water were investigated. At $50^{\circ}C$, the levels of E. sakazakii decreased one log CFU/g for 4-6 min and thereafter the levels remained stable for 20 min. At $60^{\circ}C$, inactivation by about 2 log CFU/g occurred for 20 min. Therefore, the unprocessed agricultural products might be a source of contamination for PIF when used as an ingredient after drying and pulverization. Rehydration of PIF for infant feeding with a water temperature of $60^{\circ}C$ rather than $50^{\circ}C$, as recommended by the manufacturers, may be helpful in the reduction of potential E. sakazakii risk.