• Journal of Nutrition and Health
• /
• 제37권9호
• /
• pp.763-770
• /
• 2004

This study was designed to compare the anti-carcinogenic effect of conjugated linoleic acid isomers on tumor incidence, cell proliferation and the levels of thromboxane (TX) B$_2$, prostaglandin (PG) E$_2$ and 1,2-diacylglycerol (DAG), and the related enzyme expression of cyclooxygenase (COX)-2 and protein kinase C (PKC) in colonic mucosa of 1,2-dimethy- lhydrazine (DMH) -treated rats. One hundred eight male Sprague Dawley rats were randomly divided into 3 groups depending on the types of CLA isomers, i.e. control group (no CLA contained), c9t11 group (cis-9, trans-11 CLA contained), and t10c12 group (trans-10, cis-12 CLA contained). The experimental diet was composed of protein at 20%, carbohydrate at 56.2%, and fat at 14.5% including 1.0% CLA isomers by weight. The experimental diet was fed for 30 weeks with the initiation of intramuscular injection of DMH, which was injected twice a week for 6 weeks to give total dose of 180 mg per kg body weight. Two CLA isomers (c9, t11, t10, c12) significantly reduced tumor incidence and cell proliferation by reducing the protein expression of COX-2 and PKC, and the level of TXB$_2$, PGE$_2$, and DAG in colonic mucosa. However, there was no significant difference in anti-carcinogenic effect between c9t11-CLA and t10c12-CLA.

• The Korean Journal of Physiology and Pharmacology
• /
• 제2권2호
• /
• pp.251-260
• /
• 1998

Cromakalim (BRL 34915), known as an airway smooth muscle relaxant, inhibited the releases of mediators in the antigen-induced mast cell activation. It has been suggested that cromakalim, in part, inhibited mediator releases by inhibiting the initial increase of 1,2-diacylglycerol (DAG) produced by the activation of the other phospholipase system which is different from phosphatidylcholine-phospholipase D pathway. The aim of this study is to further examine the inhibitory mechanism of cromakalim on the mediator release in the mast cell activation. Guinea pig lung mast cells were purified by using enzyme digestion and percoll density gradient. In purified mast cells prelabeled with $[^3H]PIP_2$, phospholipase C (PLC) activity was assessed by the production of $[^3H]$insitol phosphates. Protein kinase C (PKC) activity was assessed by measuring the protein phosphorylated from mast cells prelabeled with $[{\gamma}-32P]ATP$, and Phospholipase $A_2\;(PLA_2)$ activity by measuring the lyso-phosphatidylcholine produced from mast cell prelabeled with 1-palmitoyl-2-arachidonyl $phosphatidyl-[^{14}C]choline$. Histamine was assayed by fluorometric analyzer, and leukotrienes by radioimmunoassay. The PLC activity was increased by activation of the passively sensitized mast cells. This increased PLC activity was decreased by cromakalim pretreatment. The PKC activity increased by the activation of the passively sensitized mast cells was decreased by calphostin C, staurosporine and cromakalim, respectively. The $PLA_2$ activity was increased in the activated mast cells. The pretreatment of cromakalim did not significantly decrease $PLA_2$ activity. These data show that cromakalim inhibits histamine release by continuously inhibiting signal transduction processes which is mediated via PLC pathway during mast cell activation, but that cromakalim does not affect $PLA_2$ activity related to leukotriene release.

• 대한의생명과학회지
• /
• 제14권2호
• /
• pp.69-76
• /
• 2008

${\gamma}$-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system, and its actions are mediated by subtypes of GABA receptors named as $GABA_A$, $GABA_B,\;and\;GABA_C,\;GABA_A$, receptor consisting of ${\alpha},\;{\beta},\;{\gamma}\;and\;{\delta}$ subunits is a heterooligomeric ligand-gated chloride channel. This study was performed to investigate regulation of $GABA_A$ receptor by protein kinase C(PKC). Ion currents were recorded using gramicidine-perforated patch and whole cell patch clamp. mRNA encoding the subunits of PKC expressed in major pelvic ganglion (MPG) neurons was detected by using RT-PCR. The GABA-induced inward current was increased by PKC activators and decreased by PKC inhibitors, respectively. These effects were not associated with intracellular $Ca^{2+}$ and GAG (1-oleoyl-2-acetyl-sn-glycerol), a membrane permeable diacylglycerol (DAG) analogue. These results mean that the subfamily of PKC participating in activation of $GABA_A$ receptor would be an atypical PKC (aPKC). Among theses, ${\xi}$ isoform of aPKC was detected by RT-PCR. Taking together, we suggest that excitable $GABA_A$ receptor in sympathetic MPG neuron seemed to be regulated by aPKC, particular in ${\xi}$ isoform. The regulatory roles of PKC on excitatory $GABA_A$ receptors in sympathetic neurons of MPG may be an important factor to control the functional activity of various pelvic organs such as bowel movement, micturition and erection.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
• /
• pp.274-274
• /
• 1994

본 연구에서는 토끼신장 근위세뇨관 일차배양세포에서 브라디키닌의 생리작용이 phospholipase D (PLD)에 의해 매개되는지를 살펴 보기위해 PLD 효소반응의 특이한 성질인 transphosphatidylation 반응의 생성물인 phosphatidylethanol (PEth) 의 세포내 양을 측정함으로 PLD 효소의 관련성을 규명할 수 있었다. 시간경과에 따른 phosphatidic acid (PA) 및 diacylglycerol (DAG) 의 생성을 살펴본 결과 PA가 DAG보다 먼저 생성되어 최고치 (30초)에 도달하였고 DAG는 1분이후부터 5분까지 서서히 생성되는 양상을 나타내었다. 또한 0.5에서 5％까지의 에탄올 존재하에 PA 및 PE소 생성량을 비교해본 결과 에탄올량이 증가함에 따라 PA는 감소하는 반민 PEth 의 생성은 계속 증가하였다. 한편 브라디키닌 농도 변화 실험에서는 브라디키닌농도가 증가함에 따라 PA 및 PEth 둘다 생성이 증가되었다. 이러한 결과로부터 토끼신장 근위세뇨관 세포막에 존재하는 브라디키닌수용체는 브라디키닌에 의해 activation 시 PLD를 직접적으로 활성화시켜 그들의 작용을 세포내로 전달한다는 사실을 알 수 있었다. 또한 PLD 효소활성의 activator로 수용체효능 제외에 칼슘이온, protein kinase C (PKC) 등이 몇몇 다른 실험에 의해 밝혀져 있고, G protein 역시 PLD 효소 활성을 조절하는 역할이 있음이 알려졌다. calcium ionophore 및 칼슘채널길항제인 verapamil을 이용한 실험에서 우리는 브라디키닌의 PLD 활성화는 칼슘이온에 의존적인 경로 및 비의존적인 경로가 같이 존재함을 알수 있었다. 또한 브라디키닌의 PLD 활성화기전이 PKC 의존적인지를 살펴보기위해 PKC activator(PMA) 및 inhibitor (staurosporine)를 이용한 실험에서 브라디키닌은 신장세포에서 PKC를 통하여 PLD를 활성화시킴으로 신호전달을 하는 것으로 추측되었다. 마지막으로 가수분해안되는 G protein 유도체인 GTPrS 및 G protein 활성물질 NaF, 백일해독소등을 이용한 실험에서 G protein 의 PLD 조절활성을 확인할 수 있었다.

• 한국미생물·생명공학회지
• /
• 제32권1호
• /
• pp.84-90
• /
• 2004

Lipase를 사용하여 대두유와 글리세롤로부터 디글리세리드를 생산하였다. 디글리세러드의 초기 생산 속도는 리파제의 양에 크게 영향을 받았다 그러나 반응혼합물에 첨가된 리파제의 양은 평형상태의 디글리세리드의 함량에는 거의 영향을 미치지 않았다. 초기 지방산 생성속도는 0.5-2.3%의 글기세롤 수분함량에서 매우 영향을 받았으며 3.3-5.2%의 수분함량에서는 느리게 증가하였다 평형상태에서의 DG 함량은 글리세롤 수분함량 4.4%까지 서서히 증가하였으며 5.2-6.4%의 수분함량에서는 지방산함량의 증가로 인하여 오히려 감소하였다 가장 높은 디글리세리드 생산효율을 나타내는 반응온도는 $30-50^{\circ}C$이었으며 글리세롤과 트리글리세드의 몰 비율은 최종적인 디글리세리드 생산율에는 영향을 미치지 많았다. 글리세롤(GL)과 트리글리세리드(TG)의 몰 비율이 0.75:1인 경우에 높은 효소촉매반응을 나타냈으며 첨가된 글리세롤이 반응에 모두 이용될 수 있는 최적조건으로 확인되었다. 최적화된 조건에서 45%의 디글리세리드를 얻을 수 있었으며 전체 디글리세리드 중 65%는 1,3-디글리세리드였다

• Journal of Microbiology and Biotechnology
• /
• 제25권11호
• /
• pp.1827-1834
• /
• 2015

The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Site-directed mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and p-nitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipid-anchoring mechanism for DAG (L103 and F104 serve as "anchors" to the lipid interface) were proposed.

• 한국응용과학기술학회지
• /
• 제15권4호
• /
• pp.35-44
• /
• 1998

All the triacylglycerols including the molecular species having ${\Delta}^5$-unsaturated fatty acids from the seeds of Pinus Koraiensis, were split into a mixture of diacylglycerols by a Grignard reagent prepared with allyl bromide without arousing acyl chains of a glycerol moiety to migration, and were also easily partially hydrolyzed to diacylglycerols by pancreatic lipase. (S)-(+)-(1-naphthyl)ethyl urethane(NEU) derivatives of the diacylglycerol mixture derived from the triacylglycerols were fractionated into sn-1, 3-, sn-1, 2- and sn-2, 3-DG-NEU by silica-HPLC and the fatty acid composition of these fractions was analysed. $C_{18:1{\omega}9}$ is distributed evenly in the three positions of TG with $C_{18:2{\omega}6}$ mainly located in sn-2 position, while ${\Delta}^5$-unsaturated fatty acids such as ${\Delta}^{5.9}-C_{18:2}$, ${\Delta}^{5.9.12}-C_{18:3}$ and ${\Delta}^{5.11.14}-C_{20:3}$ are exclusively present in the sn-3 position. These results could be confirmed by $^{13}C$-NMR spectroscopy : the signals at $^{\delta}$173.231 ppm and $^{\delta}$172.811 ppm of the carbonyl carbon of acyl moieties indicate the presence of saturated acids and/or $C_{18:1{\omega}9}$ (oleic acid) in the ${\alpha}({\alpha}')$- or ${\beta}$- positions, and $C_{18:2{\omega}6}$ including $C_{18:1{\omega}9}$ in the ${\beta}$-position, respectively. In addition, the resonance at $^{\delta}$173.044 ppm suggested a location of ${\Delta}^5$-unsaturated fatty acid moiety in the ${\alpha}({\alpha}')$-position.

• BMB Reports
• /
• 제32권1호
• /
• pp.6-11
• /
• 1999

To understand the function of phospholipids and their fatty acid composition on the morphological changes in the amp 1-4 mutant of Arabidopsis, the mutant was compared to the wild-type Arabidopsis by TLC, HPTLC, phosphorous assay, HPLC, and GC. In the mutant, phosphatidylethanolamine (PE) was increased 5-fold and phosphatidylglycerol (PG) was decreased 1.2-fold (nmol phosphorous/g tissue). Inositol phospholipids showed a generally increased trend ranging from 1.4-to 3.0-fold (nmol inositol/g tissue). When fatty acid composition of the mutant was compared to the wild-type, linoleic (18:2) and linolenic (18:3) acids of phosphatidylcholine (PC) and PG were decreased but palmitoleic acid (16:1) and oleic acid (18:1) of PC was increased 2.5- and 2.1-fold (mol%), respectively. In galactolipids, myristic acid (14:0) of monogalactosyl-diacylglycerol (MGDG) were increased 5.8-fold (mol%). Among the inositol phospholipids, lysophosphatidylinositol (L-PI) and phosphatidylinositol 4,5-bisphosphate ($PIP_2$) showed 4-and 1.9-fold (mol%) increase of 16:1, respectively. These results suggest that the increase of PE, the decrease of PG, the increase of inositol phospholipids, and the altered fatty acid composition are related to the phenotypic changes affecting the morphological features, and might cause different physiological changes in the amp 1-4 mutant compared to wild-type Arabidopsis.

• 대한수의학회지
• /
• 제38권3호
• /
• pp.508-517
• /
• 1998

There is evidence that the sympathetic nervous system exerts a control on thyroid function via an adrenergic innervation of thyroid cells. Although it is clear that the inhibitory effects of catecholamines result from an activation of ${\alpha}_1$-adrenoceptors, the mechanisms involved in ${\alpha}_1$-stimulation are not fully understood. The effects of methoxamine and protein kinase C (PKC) activator on the release of thyroxine ($T_4$) from mouse thyroid were studied to clarify the role of PKC in the regulation of $T_4$ release in vitro. The glands were incubated in the medium, samples of the medium were assayed for $T_4$ by EIA kits. Methoxamine inhibited the TSH-stimulated $T_4$ release. This inhibition was reversed by prazosin, an ${\alpha}_1$-adrenergic antagonist. Futhermore, the inhibitory effect of methoxamine on the $T_4$ release stimulated by TSH was prevented by chloroethylclonidine, an ${\alpha}_{1b}$-adrenoceptor antagonist, but not by WB4101, an ${\alpha}_{1a}$-adrenoceptor antagonist. Also methoxamine inhibited the forskolin-, cAMP- or IBMX-stimulated $T_4$ release. These inhibition were reversed by PKC inhibitors, such as staurosporine and $H_7$. PMA, a PKC activator, completely inhibited the TSH-stimulated $T_4$ release, and its inhibition was reversed by staurosporine and $H_7$, but not by chelerythrine. R59022 (a diacylglycerol kinase inhibitor), like methoxamine, also inhibited the TSH-stimulated $T_4$ release, and its inhibition was also reversed by staurosporine. The present study suggests that methoxamine inhibition of $T_4$ release from mouse thyroid can be induced by activation of the ${\alpha}_{1b}$-adrenoceptors and that it is mediated through the ${\alpha}_1$-adrenoceptor-stimulated PKC formation.

• Asian-Australasian Journal of Animal Sciences
• /
• 제28권1호
• /
• pp.127-134
• /
• 2015

Castration induces the accumulation of body fat and deposition of intramuscular fat in Korean cattle, resulting in improved beef quality. However, little is known about the metabolic adaptations in the liver following castration. To understand changes in lipid metabolism following castration, hepatic expression levels of lipid metabolism genes were compared between Korean bulls and steers. Steers had higher (p<0.001) hepatic lipids contents and higher (p<0.01) mRNA levels of lipogenic acetyl-CoA carboxylase. This differential gene expression may, in part, contribute to increased hepatic lipid content following the castration of bulls. However, we found no differences in the hepatic expression levels of genes related to triglyceride synthesis (mitochondrial glycerol-3-phosphate acyltransferase, diacylglycerol O-acyltransferase 1 and 2) and fatty acid (FA) oxidation (carnitine palmitoyltransferase 1A, C-4 to C-12 straight chain acyl-CoA dehydrogenase, very long chain acyl-CoA dehydrogenase) between bulls and steers. No differences in gene expression for very-low-density lipoprotein (VLDL) secretion, including apolipoprotein B mRNA and microsomal triglyceride transfer protein (MTTP) protein, were observed in the liver although MTTP mRNA levels were higher in steers compared to bulls. In conclusion, FA synthesis may contribute to increased hepatic lipid deposition in steers following castration. However, hepatic lipid metabolism, including triglyceride synthesis, FA oxidation, and VLDL secretion, was not significantly altered by castration. Our results suggest that hepatic lipid metabolism does not significantly contribute to increased body fat deposition in steers following castration.