• BMB Reports
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• v.39 no.6
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• pp.717-721
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• 2006

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial pre-apo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.

• BMB Reports
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• v.57 no.10
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• pp.447-452
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• 2024

The heterotrimeric molecular motor kinesin-2 is involved in the microtubule-dependent transport of intracellular cargo. It consists of two distinct motor subunits (KIF3A, and KIF3B) and a non-motor subunit, kinesin-associated protein 3 (KAP3). The cargo-binding domain (CBD) at the carboxyl (C)-terminus of KIF3s plays an important role in the interaction with several different binding proteins. To identify the binding proteins for heterotrimeric kinesin-2, we performed a yeast two-hybrid screen and found a new interaction with Disables-1 (Dab1), the intracellular adaptor protein of reelin receptors. Dab1 bound to the CBD of KIF3A, but did not interact with the C-terminal domain of KIF3B, KIF5B, KIF17 or KAP3. The phosphotyrosine binding (PTB) domain-containing region of Dab1 is essential for the interaction with KIF3A. KIF3A interacted with GST-Dab1, and GST-CaMKIIα , but did not interact with GST-apolipoprotein E receptor 2 (ApoER2)-C or with GST alone. When co-expressed in HEK-293T cells, Dab1 co-precipitated with KIF3A, but not with KIF5B. Dab1 and KIF3A were co-localized in cultured cells. We also identified deduced cell surface expression of ApoER2 in KIF3A dominant-negative cells. These results suggest that the KIF3A plays a role in the intracellular trafficking of ApoER2 to the cell surface.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.236-244
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• 2006

This study was designed to determine the effects of daidzein (DE) on hepatic lipid metabolism in chicks fed with low protein (LP) diet based on casein. In experiment 1, the male chicks were fed with one of the three levels of dietary protein containing 10.95%, 21.9% and 43.8% protein content for 2 days. In experiment 2, the chicks were fed one of the three levels of protein with or without DE at 1,000 mg/kg diet for 2 days. Experiment 3 was conducted to compare DE (LP+DE) with estradiol (LP+E2) in chicks fed with LP diet for 7 days. Plasma lipid profiles, hepatic lipid profiles, activities of hepatic malic enzyme and isocitrate dehydrogenase (ICDH) were measured. Transcriptions of hepatic fatty acid synthase, apolipoprotein-B (APO-B), and fructose bisphosphatase mRNA were measured by RT-PCR. Increasing dietary protein levels markedly decreased the concentrations of plasma triglycerides, hepatic total lipids, hepatic TG, and the mRNA transcriptions while the increased dietary protein levels increased hepatic ICDH activities in experiment 1. In experiment 2, the effects of dietary protein levels on blood and hepatic lipid content were more prominent than those of the additional DE. Interestingly, plasma TG levels were affected by DE supplementation (p<0.05). In experiment 3, DE inhibited APO-B mRNA expressions and stimulated the accumulation of lipid in the liver through mechanisms different from E2. In this study, we demonstrate that DE has beneficial effects on blood lipid profiles, but that it inhibits APO-B mRNA transcription and aggravates the fatty liver induced by LP diet in chicks.

• Proceedings of the KSCN Conference
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• 2001.12a
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• pp.975.1-975
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• 2001

• Animal cells and systems
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• v.9 no.3
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• pp.145-152
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• 2005

A retrograde tracer, WGA-apo-HRP-gold, was injected into midline thalamic nuclei and subsequently orexin-A immunostaining was performed on the tuberal region of the hypothalamus in order to investigate orexinergic projections to the midline thalamus. Injection site was targeted within one specific region, i.e., paraventricular, centromedian, rhomboid, reuniens, or intermediodorsal nucleus, but it proved to be either one or a combination of these thalamic nuclei. The distribution of WG/orexin-double-labeled neurons exhibited a general pattern in that the majority of labeled cells were observed within the ventral portion of the lateral hypothalamus as well as the perifornical nucleus (PeF). A small number of double-labeled cells were also observed at the dorsomedial nucleus, the area dorsal to the PeF, dorsal portion of the lateral hypothalamus, and the posterior hypothalamus. These orexin-immunoreactive neurons might have wake-related influences over a variety of functions related with midline thalamic nuclei, which include autonomic control, associative cortical functions, and limbic regulation.

• Journal of the Optical Society of Korea
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• v.11 no.1
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• pp.26-33
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• 2007

We demonstrate the capability of differential-phase optical coherence tomography (DP-OCT) to detect superparamagnetic iron oxide (SPIO) nanoparticles taken up by liver parenchymeal macrophages (Kupffer cells). We apply an external time-varying high-intensity focused magnetic field. Our experiments demonstrate a novel diagnostic modality to detect macrophages that have taken up SPIO nanoparticles. Magnetic force acting on the nanoparticles was varied by applying a sinusoidal current to a solenoid containing a conical iron core that substantially increased and focused the magnetic field strength ($B_{max}$ = 2 Tesla). $ApoE^{-/-}$ mice were sacrificed 2 days post intravenous injections of different SPIO doses (1.0, and 0.1 mmol Fe/kg body weight). Livers of $ApoE^{-/-}$ mice with and without injection of SPIO nanoparticles were investigated using DP-OCT, which detects tissue movement with nanometer resolution. Frequency response of iron-laden liver movement was twice the stimulus frequency. Movement was not observed in livers of control mice. Results of our experiments indicate DP-OCT is a candidate methodology to detect tissue based macrophages containing SPIO nanoparticles excited by an external focused magnetic field.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1331-1341
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• 2001

Dairy cows are prone to fatty liver during the time of periparturient. Despite of the extensive studies, etiology and solutions for fatty liver are still not well known.The liver synthesizes triglycerides (TG) using precursors from bloodstream and secretes TG in form of very low density lipoprotein (VLDL) into bloodstream for the utilization by peripheral tissues. When the amount of TG synthesis exceeds the amount of secretion in VLDL-TG, TG accumulation within the liver occurs. Hepatic VLDL assembly and secretion involve multi-biochemical events.The availabilities of apolipoprotein B (apoB), E (apoE), microsomal triglyceride transfer protein (MTP) and soluble low density lipoprotein (LDL) receptor are now believed to be some of the main regulators for hepatic VLDL assembly and secretion. Studies in transgenic animals show that overexpression of these proteins stimulates VLDL production and secretion, which provides a possibility for alleviating bovine fatty liver by gene therapy. However, many problems remain to be solved to attain this goal. This review focuses on the molecular mechanisms of hepatic VLDL assembly and secretion, and the possibilities and problems of applying the knowledges to solve bovine fatty liver by gene therapy.

• Journal of Korean Medicine Rehabilitation
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• v.24 no.2
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• pp.1-13
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• 2014

Objectives The purpose of this study was to investigate the effects of Cheunggihwadamhwan (CGHDH) extract on lowering lipid, antioxidation and production of proinflammatory cytokines in rats fed on high fat diet. Methods 40 Male Sprague-Dawley rats were fed on high fat diet for 8 weeks and 32 rats (above 400 g) were randomly divided into 4 groups (8 mice in each group) : control group, 100 mg/Kg CGHDH group, 200 mg/Kg CGHDH group, 300 mg/Kg CGHDH group. We fed a control group of rats a basal diet and administered normal saline(100 mg/kg, 1 time/1 day) for 4 weeks. And We fed each experimental group of rats basal diet and administered an extract of Cheunggihwadamhwan extracts (100 mg/kg, 200 mg/kg, 300 mg/kg, 1 time/1 day) for 4 weeks. At the end of the experiment, the rats were sacrificed to determine their chemical composition. We measured lipid in plasma and liver, concentration of proinflammatory cytokines, antioxidative activity and gene expression. The gene expression level was investigated by the way of reverse transcription-polymerase chain reaction (RT-PCR). Results 1. Concentration of plasma FFA, plasma TG, plasma total cholesterol and plasma LDL-cholesterol showed a significant decrement in Cheunggihwadamhwan groups. However, concentration of plasma HDL-cholesterol showed a significant increment in 200, 300 mg/kg Cheunggihwadamhwan groups. 2. Concentration of liver total cholesterol and liver TG showed a significant decrement in Cheunggihwadamhwan groups. 3. Concentration of plasma TBARS showed a significant decrement in all Cheunggihwadamhwan groups. Concentration of liver TBARS showed a significant decrement in 200, 300 mg/kg Cheunggihwadamhwan groups. Concentration of liver GSH-Px, SOD and CAT showed a tendency to decrease in all Cheunggihwadamhwan groups. 4. Concentration of plasma IL-$1{\beta}$, plasma IL-6, TNF-$\alpha$ and NO, showed a tendency to decrease in all Cheunggihwadamhwan groups. Concentration of plasma IL-10 showed a tendency to increase in all Cheunggihwadamhwan groups. 5. In the analysis of reverse transcription-polymerase chain reaction (RT-PCR), the gene expression of Apo-B and Apo-E in the Cheunggihwadamhwan groups showed a low expression than that of control group. The ratio of Apo-B expression per $\beta$-actin expression in the showed a significant decrement in all Cheunggihwadamhwan groups. The ratio of Apo-E expression per $\beta$-actin expression in the showed a significant decrement in 300 mg/kg Cheunggihwadamhwan groups. Conclusions According to this study, the extract of Cheunggihwadamhwan showed a positive effect of lowering lipid, antioxidation and a control of producing proinflammatory cytokines.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.427-433
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• 2008

The cephabacins produced by Lysobacter lactamgenus are ${\beta}$-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, ${\beta}$-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of $His_6$-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-$^3H$-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with $^{14}C$-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.

• Preventive Nutrition and Food Science
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• v.2 no.2
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• pp.121-128
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• 1997

this study was conducted to examine the atherogenic effect of high cholesterol diet (experimental diet) that influences changes of lipoprotein cholesterol metabolism and arterial wall. Seven NewZealand white rabbits were fed control diet, an the other 7 rabbits 2% cholesterol diet for 10 weeks. Results obtained from this study are as follows: 1) High cholesterol diet resulted in a gradual increase of plasma total cholesterol level, reaching upto 1422 mg/dl at the seventh week. 2) CETP (cholesteryl ester transfer protein) activity was significantly higher in high cholesterol group (64.9% at the 7th week) than control group (49.3% at the 7th week) during most of the experimental period except the 6th week. 3) The cholesterol supplementation induced fatty liver and a decrease of hepatic HMG-CoA reductase activities (2.1 moles vs. 0.3nmoles) compared to control group. 4) Bands of apo B-100 and apo E in plasma lipoprotein were thicker in high cholesterol-fed animals tan control animals as visualized by SDS-PAGE. 5) Oxidizability of plasma lipoproteins measured in vitro was greater in high cholesterol group tan control group, but vitamin E level higher in control group. 6) he effect of cholesterol feeding for 10 weeks also led to early fatty streaks in aortic intima. High cholesterol feeding was atherogenic to rabbits, an this seems to be mediated through elevated CETP activities that regulate plasma HDL cholesterol level and decrease an efficiency of reverse cholesterol transport in lipoprotein cholesterol metabolism. The enhanced oxidizability of plasma lipoproteins and lowered vitamin E level may also contribute to the formation of faaty streaks in aorta of cholesterol-fed rabbits.