• Analytical Science and Technology
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• v.7 no.2
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• pp.213-224
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• 1994

Several isotopomers of cyclooctanone were prepared by selective deuterium substitution. Intrinsic isotope effects on $^{13}C$ NMR chemical shifts of these isotopomers were investigated systematically at low temperature. These istope effects were discussed in relation to the preferred boat-chair conformation of cyclooctanone. Deuterium isotope effects on NMR chemical shifts have been known for a long time. Especially in a conformationally mobile molecule, isotope perturbation could affect NMR signals through a combination of isotope effects on equilibria and intrinsic effects. The distinction between intrinsic and nonintrinsic effects is quite difficult at ambient temperature due to involvement of both equilibrium and intrinsic isotope effects. However if equilibria between possible conformers of cyclooctanone are slowed down enough on the NMR time scale by lowering temperature, it should be possible to measure intrinsic isotope shifts from the separated signals at low temperature. $^{13}C$ NMR has been successfully utilized in the study on molecular conformation in solution when one deals with stable conformers or molecules were rapid interconversion occurs at ambient temperature. The study of dynamic processes in general requires analysis of spectra at several temperature. Anet et al. did $^1H$ NMR study of cyclooctanone at low temperature to freeze out a stable conformation, but were not able initially to deduce which conformation was stable because of the complexity of alkyl region in the $^1H$ NMR spectrum. They also reported the $^1H$ and $^{13}C$ NMR spectra of the $C_9-C_{16}$ cycloalkanones with changing temperature from $-80^{\circ}C$ to $-170^{\circ}C$, but they did not report a variable temperature $^{13}C$ NMR study of cyclooctanone. For the analysis of the intrinsic isotope effect with relation to cylooctanone conformation, $^{13}C$ NMR spectra are obtained in the present work at low temperatures (up to $-150^{\circ}C$) in order to find the chemical shifts at the temperature at which the dynamic process can be "frozen-out" on the NMR time scale and cyclooctanone can be observed as a stable conformation. Both the ring inversion and pseudorotational processes must be "frozen-out" in order to see separate resonances for all eight carbons in cyclooctanone. In contrast to $^1H$ spectra, slowing down just the ring inversion process has no apparent effects on the $^{13}C$ spectra because exchange of environments within the pairs of methylene carbons can still occur by the pseudorotational process. Several isotopomers of cyclooctanone were prepared by selective deuterium substitution (fig. 1) : complete deuterium labeling at C-2 and C-8 positions gave cyclooctanone-2, 2, 8, $8-D_4$ : complete labeling at C-2 and C-7 positions afforded the 2, 2, 7, $7-D_4$ isotopomer : di-deuteration at C-3 gave the 3, $3-D_2$ isotopomer : mono-deuteration provided cyclooctanone-2-D, 4-D and 5-D isotopomers : and partial deuteration on the C-2 and C-8 position, with a chiral and difunctional case catalyst, gave the trans-2, $8-D_2$ isotopomer. These isotopomer were investigated systematically in relation with cyclooctanone conformation and intrinsic isotope effects on $^{13}C$ NMR chemical shifts at low temperature. The determination of the intrinsic effects could help in the analysis of the more complex effects at higher temperature. For quantitative analysis of intrinsic isotope effects, the $^{13}C$ NMR spectrum has been obtained for a mixture of the labeled and unlabeled compounds because the signal separations are very small.

• Journal of the Korean Chemical Society
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• v.51 no.1
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• pp.43-50
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• 2007

Liposomes having particle size from several tens to hundreds nanometers are efficient carriers for injectable drug delivery. Enhancement of liposome stability in bloodstream has been studied because of its relatively short circulation time and fast clearance from human body by reticuloendothelial system (RES) in blood vessel. In this study, new disaccharide-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) derivatives in which lactose or sucrose as the disaccharide molecule was conjugated covalently to DSPE were synthesized. Liposomes of which surface had disaccharide molecules were prepared by incorporating the disaccharide-DSPE into liposomes as one of their lipid components. Particle size of the prepared liposomes was approximately 100 nm. The liposomes of which surface were modified with the disaccharide-DSPE showed -25 mV of zeta potential value due to the presence of hydroxyl groups on their surface, while the unmodified control liposomes showed -10 mV of zeta potential value. Loading efficiency of model drug, doxorubicin, into liposomes was about 90%. Stability of the disaccharide-modified liposomes in vitro was evaluated by monitoring the amount of protein adsorption and particle size of the liposomes in serum. Disaccharide-modified liposomes were more stable in serum than unmodified control liposomes or polyethyleneglycol (PEG)-modified liposomes due to less adsorption of serum protein and hence less increase of their particle size. The liposomes of which surface was modified with disaccharide-DSPE conjugate can be used as long-circulating carriers for drugs having high toxicity or short half-life time due to their enhanced stability in blood circulatory system.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.39-47
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• 2015

Secondary metabolism in actinomycetes has been known to be controlled by a small molecule, ${\gamma}$-butyrolactone autoregulator, the binding of which to each corresponding receptor leads to the regulation of the transcriptional expression of the secondary metabolites. We expected that expression of an autoregulator receptor or a pleiotropic regulator in a non-host was to be gained insight of effective production of new metabolic materials. In order to study the function of the receptor protein (seaR), which is isolated from Saccharopolyspora erythraea, we introduced the seaR gene to Streptomyces coelicolor A3(2) as host strains. An effective transformation procedure for S. coelicolor A3(2) was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP and $ermEp^*$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. coelicolor A3(2) by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Exconjugant of S. coelicolor A3(2) containing the seaR gene was confirmed by PCR and transcriptional expression of the seaR gene in the transformant was analyzed by RT-PCR. In case of S. coelicolor A3(2), a phenotype microarray was used to analyze the phenotype of transformant compared with wild type by seaR expression. After that, in order to confirm the accuracy of the results obtained from the phenotype microarray, an antimicrobial susceptibility test was carried out. This test indicated that sensitivity of the transformant was higher than wild type in tetracycline case. These results indicated that some biosynthesis genes or resistance genes for tetracycline biosynthesis in transformant might be repressed by seaR expression. Therefore, subsequent experiments, analysis of transcriptional pattern of genes for tetracycline production or resistance, are needed to confirm whether biosynthesis genes or resistance genes for tetracycline are repressed or not.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.3 s.47
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• pp.393-397
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• 2004

L-Carnitine $({\beta}-hydroxy-{\gamma}-trimethyl-ammoniumbutyric{\;}acid)$ is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-carnitine, we investigated the effects of in vitro matrix-metalloproteinase (MMP) inhibition and activity and expression of UYA-induced MMPs in human skin fibroblasts. Also, we studied to develop as anti-aging cosmetics with L-carnitine. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. ELISA (enzyme linked immune sorbent assay), gelatin-substrate zymography, RT-PCR ELISA techniques were used for the effects of L-carnitine on MMP expression, activity, and MMP mRNA expression in UVA irradiated fibroblast $(5\;J/cm^2)$, respectively. In addition, we performed clinical study with L-carnitine cream. L-carnitine inhibited the activities of MMP-1 in a dose-dependent manner and the $IC_{50}$ values calculated from semi-log plots were 2.45 mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP-1, 2 expression was reduced $43\%,\;53\%$ by treated with L-carnitine at 1.25 mM, and MMP-1 mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibit the MMP activity, and regulate MMP expression in protein and mRNA level. The results of clinical study showed that $1.0\%$ L-carnitine treated group reduced wrinkle significantly compared with placebo treated group (P<0.05). All these results suggest that L-carnitine may be useful as new anti-aging cosmetics for protection against UVA induced Mm expression and activity.

• Applied Biological Chemistry
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• v.10
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• pp.1-13
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• 1968

Phytin is a salt(mainly calcium and magnesium) of phytic acid and its purity and molecular formula can be determined by assaying the contents of phosporus, calcium and magnesium in phytin. In order to devise a new method for the quantitative analysis of the three elements in phytin, the chelatometric method was developed as follows: 1) As the pretreatment for phytin analysis, it was ashfied st $550{\sim}600^{\circ}C$ in the presence of concentrated nitric acid. This dry process is more accurate than the wet process. 2) Phosphorus, calcium and megnesium were analyzed by the conventional and the new method described here, for the phytin sample decomposed by the dry process. The ashfied phytin solution in hydrochloric acid was partitioned into cation and anion fractions by means of a ration exchange resin. A portion of the ration fraction was adjusted to pH 7.0, followed by readjustment to pH 10 and titrated with standard EDTA solution using the BT [Eriochrome black T] indicator to obtain the combined value of calcium and magnesium. Another portion of the ration fraction was made to pH 7.0, and a small volume of standard EDTA solution was added to it. pH was adjusted to $12{\sim}13$ with 8 N KOH and it was titrate by a standard EDTA solution in the presence of N-N[2-Hydroxy-1-(2-hydroxy-4-sulfo-1-naphytate)-3-naphthoic acid] diluted powder indicator in order to obtain the calcium content. Magnesium content was calculated from the difference between the two values. From the anion fraction the magnesium ammonium phosphate precipitate was obtained. The precipitate was dissolved in hydrochloric acid, and a standard EDTA solution was added to it. The solution was adjusted to pH 7.0 and then readjusted to pH 10.0 by a buffer solution and titrated with a standard magnesium sulfate solution in the presence of BT indicator to obtain the phosphorus content. The analytical data for phosphorus, calcium and magnesium were 98.9%, 97.1% and 99.1% respectively, in reference to the theoretical values for the formula $C_6H_6O_{24}P_6Mg_4CaNa_2{\cdot}5H_2O$. Statical analysis indicated a good coincidence of the theoretical and experimental values. On the other hand, the observed values for the three elements by the conventional method were 92.4%, 86.8% and 93.8%, respectively, revealing a remarkable difference from the theoretical. 3) When sodium phytate was admixed with starch and subjected to the analysis of phosphorus, calcium and magnesium by the chelatometric method, their recovery was almost 100% 4) In order to confirm the accuracy of this method, phytic acid was reacted with calcium chloride and magnesium chloride in the molar ratio of phytic: calcium chloride: magnesium chloride=1 : 5 : 20 to obtain sodium phytate containing one calcium atom and four magnesium atoms per molecule of sodium phytate. The analytical data for phosporus, calcium and magnesium were coincident with those as determine d by the aforementioned method. The new method employing the dry process, ion exchange resin and chelatometric assay of phosphorus, calcium and magnesium is considered accurate and rapid for the determination of phytin.

• Tuberculosis and Respiratory Diseases
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• v.43 no.1
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• pp.75-87
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• 1996

Background: Lung cancer is the second most frequent malignancy in man in Korea. Surgery is the best treatment modality for non-small cell lung cancer, but most patients were presented in far advanced stage. So radiation therapy(RT) with or without chemotherapy is the next choice and radiation-induced pneumonitis and pulmonary fibrosis is the major limiting factor for the curative RT. Radiation pneumonitis is manifested with fever, cough and dyspnea, 2~3 months after the termination of radiotherpy. Chest X ray shows infiltration, typically limited to the radiation field, but occasionally bilateral infiltration was reported. Also Gibson et al reported that BAL lymphocytosis was found in both lungs, even though the radiation was confined to one lung. The aim of this study is to investigate the change of adhesion molecules expression on BAL cells and serum soluble ICAM-1(sICAM-1) level after the RT and its relationship to the development of radiation pneumonitis. The second aim is to confirm the bilaterality of change of BAL cell pattern and adhesion molecule expression. Subjects: BAL and the measurement of sICAM level in serum and BALF were done on 29 patients with lung cancer who received RT with curative intention. The BAL was done before the RT in 16 patients and 1~2 month after RT in 18 patients. 5 patients performed BAL before and after RT. Result: Clinically significant radiation pneumonitis developed in 7 patients. After RT, total cell count in BAL was significantly increased from $(20.2{\pm}10.2){\times}10^6\;cells/ml$ to $(35.3{\pm}21.6){\times}10^6\;cells/ml$ (p=0.0344) and %lymphocyte was also increased from $5.3{\pm}4.2%$ to $39.6{\pm}23.4%$ (p=0.0001) in all patient group. There was no difference between ipsilateral and contraleteral side to RT, and between the patients with and without radiation-pneumonitis. In whole patient group, the level of sICAM-1 showed no significant change after RT(in serum: $378{\pm}148$, $411{\pm}150\;ng/ml$, BALF: $20.2{\pm}12.2$, $45.1{\pm}34.8\;ng/ml$, respectively), but there was a significant difference between the patients with pneumonitis and without pneumonitis (serum: $505{\pm}164$ vs $345{\pm}102\;ng/ml$, p=0.0253, BALF: $67.9{\pm}36.3$ vs $25.2{\pm}17.9\;ng/ml$, p=0.0112). The expression of ICAM-1 on alveolar macrophages (AM) tends to increase after RT (RMFI: from $1.28{\pm}0.479$ to $1.63{\pm}0.539$, p=0.0605), but it was significantly high in patients with pneumonitis ($2.10{\pm}0.390$) compared to the patients without pneumonitis ($1.28{\pm}0.31$, p=0.0002). ICAM-1 expression on lymphocytes and CD 18 (${\beta}2$-integrin) expression tended to be high in the patients with pneumonitis but the difference was statiastically not significant. Conclusion: Subclinical alveolitis on the basis of BAL finding developed bilaterally in all patients after RT. But clinically significant pneumonitis occurred in much smaller fraction and the ICAM-1 expression on AM and the sICAM-1 level in serum were good indicator of it.

• Journal of the Korean Applied Science and Technology
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• v.18 no.3
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• pp.197-213
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• 2001

Conjugate trienoic acids (CTA) occurred in triacylglycerols (TGs) of the seed oils of Trichosanthes kirilowii, Momordica charantia and Aleurites fordii, and they were easily converted to their methyl esters in a mixture of sodium methoxide-methanol without any structural destruction. The main fatty acids in triacylglycerol (TG) fraction of the seed oils of Trichosanthes kirilowii are $C_{18:2{\omega}6}$ (32.2 mol %), $C_{18:3{\;}9c.11t,13c}$ (38.0 mol %) and $C_{18:1{\omega}9}$ (11.8 mol %), followed with $C_{16:0}$ (4.8 mol %) and $C_{18:0}$ (3.1 mol %). The TG fraction was resolved into 20 TG molecular species according to the partition number (PN) by reversed-phase (RP)-HPLC. The main TG species were $DT_{c2}$, $MDT_{c}$ and $D_{2}T_{c}$, of which amounts reached 63 mol % of total TG molecular species. The TG sample was fractionated into 11 fractions according to the number of double bond in the molecule by $Ag^{+}-HPLC$ and the species of $DT_{c2}$, $MDT_{c}$ and $D_{2}T_{c}$ were also eluted as main components. The TG species containing CTA showed unusual behaviours in the order of elution by HPLC ; first, TG moleular species of $DT_{c2}$ (D; dienoic acid, $T_{c}$; punicic acid, $T_{ci}$; ${\alpha}-eleostearic$ acid, M ; monoenoic acid, $S_{t}$; stearic acid) was eluted earlier than $Mt_{c2}$, although they have the same PN number of 40, and, secondly, the species of $DT_{ci2}$ with eight double bonds was eluted earlier than that of $D_2T_{ci}$ with seven double bonds. Intact TG of the seed oils of Momordica charantia contained mainly fatty acids such as $C_{18:3{\omega}9c,11t,13t}$ (57.7 mol %), $C_{18:1{\omega}9}$ (17.4 mol %), $C_{18:0}$ (12.3 mol %) and $C_{18:2{\omega}6}$ (10.6 mol %), and was classified into 13 fractions by RP-HPLC. The main TG species were as follows ; $MT_{ci2}$ [$(C_{18:1{\omega}9})(C_{18:3\;9c,11t,13t})_{2}$, 39.1 mol %] and $S_{t}T_{ci2}$ [$(C_{18:0})(C_{18:3\;9c,11t,13t})_2$, 33.9 mol %] comprising about 73 mol % of total TG species, accompanied by $DT_{ci2}$ [$(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13t})_{2}$, 7.3 mol %], $D_{2}T_{ci}$ [$ (C_{18:2{\omega}6})_{2}(C_{18:3\;9c,11t,13t})$, 3.6 mol %] and $MDT_{ci}$ [$(C_{18:1{\omega}9})(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13t})$, 3.5 mol %]. Simple TG species of $T_{ci3}$ [$(C_{18:3\;9c,11t,13t})_3]$ was present in a small amount of 1.4 mol %, but other simple TG species were not detected. The TG was also resolved into 11 fractions according to the number of double bond by $Ag^{+}-HPLC$, and the species were mainly occupied by $MT_{ci2}$ [$(C_{18:1{\omega}9})(C_{18:3\;9c,11t,13t})_{2}$, 39.4 mol %] and $S_tT-{ci2}$ [$(C_{18:0})(C_{18:3\;9c,11t,13t})_{2}$, 35.4 mol %] $DT_{ci2}$ species with eight double bonds was also developed faster than $D_2T_{ci}$ one with seven double bonds as indicated in the analysis of TG of the seed oils of T. kirilowii, and $MT_{ci2}$ species with cis, trans, trans-configurated double bond was eluted earlier than $MT_{c2}$ species with cis, trans, cis-configurated double bond. The main components of fatty acid in total TG fraction isolated from the seed oils of of Aleurites fordii were in the following order ; $C_{18:3\;9c,11t,13t}$ (81.2 mol %)> $C_{18:2{\omega}6}$ (8.5 mol %)> $C_{18:1{\omega}9}$ (5.4 mol %)$. With resolution of the TG by RP-HPLC, eight fractions such as $T_{ci3}$, $Dt_{ci2}$, $D_{2}T_{ci}$, $MT_{ci2}$, $PT_{ci2}$ (P; palmitic acid), $PMT_{ci}$, $PDT_{ci}$ and $S_{t}T_{ci2}$ ($S_{t}$; stearic acid) were isolated, respectively. TG species of $T_{ci3}$ [$(C_{18:3\;9c,11t,13t})_{3}$, 54.2 mol %], $DT_{ci2}$ [$(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13t})_{2}$, 15.0 mol %] and $MT_{ci2}$ [$(C_{18:1{\omega}9})(C_{18:3 9c,11t,13t})_{2}$, 14.8 mol %] were present as main species.