• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.897-905
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• 2003

Phospholipase D (PLD) and ADP-ribosylation factor (ARF) were partially purified on a series of column chromatography, and their biochemical properties were characterized to understand the regulatory mechanism of PLD activation by ARF protein in the antigen-induced immune responses in guinea pigs. Heparin Sepharose and high-Q Sepharose column chromatographies were used for the purification of PLD, and Sephadex G-25, DEAE Sephacel, Source 15 PHE (HIC), Superdex-75, and Uno-Q column chromatographies were used for the purification of ARF. The purified PLD and ARF proteins were identified with anti-rabbit PLD- or ARF-specific antibodies, showing about 64 or 85 kDa for the molecular mass of PLD and 29 or 35 kDa for the sizes of ARF. Partial cDNA of ARF3 was cloned by RT-PCR in guinea pig lung tissue and its nucleotides and amino acids were sequenced. Guinea pig ARF3 showed 92% of nucleotides sequence identity and 100% of amino acid sequence homology with human ARF3. The ARF-regulated PLD activity was measured in the oleate or ARFs-containing mixed lipid vesicles. The purified and recombinant ARF (rARF) activities were assessed with the $GTP{\gamma}S$ binding assay. The PLD activity was induced by oleate in a dose-dependent manner. The purified ARF and recombinant ARF3 increased PLD activity in guinea pig lung tissues. These data show that the activity of membrane-bound PLD can be regulated by the cytosolic ARF proteins, suggesting that ARF proteins in guinea pig lung can act as a regulatory factor in controlling the PLD activity in allergic reaction.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.673-679
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• 2003

Glutamine and urea, abundant in body fluids or plasma, yield net ammonium ions upon hydrolysis by ${\gamma}-glutamyl$ transpeptidase (${\gamma}-GTP$) and urease, respectively, and these two enzymes are largely produced from Helicobacter pylori. To investigate bacterial potential of ammonium production, we first quantified those in whole-cell systems and found that the relative ratio of their amounts varied greatly, especially with pH values and the cell's aging. During the H. pylori cultivation, the ratio appeared to be inversely proportional to each other, showing a progressive increase of the ${\gamma}-GTP$ with decreasing of the urease. Under the urease-defective conditions due to low pH or coccoids, the bacterial cells still possessed a considerable amount of ${\gamma}-GTP$, which was found exclusively in the external compartment, therefore, the cell's ammonium production was found to be solely dependent upon glutamine, and the external ammonium concentration was constant without any contribution of urea concentration. Such ammonium constancy would definitely have an adverse effect on the host, because of its absolute requirement for vacuolar degeneration by H. pylori VacA, maximized at approximately 10 mM $NH_4Cl$. It was also found that, by using the metal-saturated membrane vesicles, ammonium ions were likely to be involved in the pH-dependent cation-flux across the H. pylori membrane, where the role of ${\gamma}-GTP$ in ammonium homeostasis around cells was suggested, especially under the hostile conditions against H. pylori.

• Korean Journal of Medicinal Crop Science
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• v.7 no.2
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• pp.129-137
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• 1999

The structure of amyloplasts and intercellular transport in the old and new bulbs of Frjtillaria pallidiflora were observed by means of electron microscope. The structure of internal membrane system was different between new and old amyloplasts. The active intercellular transport was observed in both new and old bulbs. The phenomena of encytosis and exocytosis always could be found in the cell membrane, and plasmodesmata established a symplasmic pathway for intercellular transport. Groups of vesicles often located at the ends of plasmodesmata, showing that they participated in the intercellular transport. These results laid a foundation for the further study on the mechanism of growth and development in Fritillaria pallidiflora.

• BMB Reports
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• v.33 no.3
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• pp.221-227
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• 2000

The distribution of barbiturates in the model membranes of total lipids (SPMVTL) and total phospholipids (SPMVPL) extracted from synaptosomal plasma membrane vesicles was determined by employing a fluorescent probe technique. The two fluorescent probes 2-(9-anthroyl)stearic acid and 12-(9-anthroyl)stearic acid were utilized as probes for the surface and the hydrocarbon interior of the outer monolayer of the SPMVTL and SPMVPL, respectively. The Stern-Volmer equation of fluorescent quenching was modified to calculate the relative distribution. The analysis of preferential quenching of these probes by barbiturates indicates that pentobarbital, hexobarbital, amobarbital and phenobarbital are predominantly distributed on the surface area, while thiopental sodium has an accessibility to the hydrocarbon interior of the outer monolayer of the SPMVTL and SPMVPL. From these results, it is strongly suggested that the more effective penetration into the hydrocarbon interior of the outer monolayer of the membrane lipid bilayer could result in a higher general anesthetic activity.

• Applied Microscopy
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• v.35 no.4
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• pp.74-83
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• 2005

The ontogeny of pollen wall in Panax ginseng was studied with transmission and scanning electron microscopy from early tetrad stage until pollen maturity. Initial indication of exine development is undulation of plasma membrane for the preparation of bacular mound. The first recognizable structure of the pollen wall is the cellulosic primexine which is formed outside of the plasma membrane while microspore tetrads are still surrounded by callose wall. As development proceeds, foot-layer and baculum differentiation, callose dissolution and exine formation were progressed. During this process, sporopollenin is deposited into the exine, and then endexine development was followed. The intine, innermost pollen wall layer, is developing form hypertrophic Golgi vesicles. The thickness of exine is very even on all along the pollen wall, but intine thickness of apertural region is thicker than that of nonapertural region. Mature pollen of ginseng is $20{\mu}m$ in size, tricolpate and shows fine reticulate sculpturing.

• Molecules and Cells
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• v.22 no.2
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• pp.210-219
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• 2006

Dynamin-related protein 2A (AtDRP2A, formally ADL6), a member of the dynamin family, is critical for protein trafficking from the TGN to the central vacuole. However, the mechanism controlling its activity is not well understood in plant cells. We isolated Arabidopsis sec13 homolog1 (AtSeh1) that interacts with AtDRP2A by a yeast two-hybrid screening. AtSeh1 has four WD40 motifs and amino acid sequence homology to Sec13, a component of COPII vesicles. Coimmunoprecipitation and protein pull-down experiments demonstrated specific interaction between AtSeh1 and AtDRP2A. AtSeh1 bound to the pleckstrin homology domain of AtDRP2A in competition with the C-terminal domain of the latter, and this resulted in inhibition of the interaction between AtDRP2A and PtdIns3P in vitro. AtSeh1 localized to multiple locations: the nucleus, the prevacuolar compartment and the Golgi complex. Based on these results we propose that AtSeh1 plays a role in regulating cycling of AtDRP2A between membrane-bound and soluble forms.

• Polymer(Korea)
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• v.35 no.5
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• pp.419-423
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• 2011

Chitosan is a natural polymer derived from chitin that has been widely used as a dietary supplement and in a variety of pharmacological and biomedical applications. In addition, water-soluble chitosan has been used to enhance the stability of chitosan in water and reduce cytotoxic activity induced by acetic acid. In this study, the antibiotic activity and mechanism of low molecular weight water-soluble chitosan (LMWSC; MW1, MW3, MW5, and MW10) were examined in pathogenic bacteria cells and vesicles containing bacterial membrane lipids. MW10 displayed potent antibacterial activity against pathogenic bacteria strains and no cytotoxicity against mammalian cells. In addition, the degree of calcein leakage was examined as a function of lipid composition (PE/PG=7/3 w/w). The results of these experiments demonstrated that MW10 promoted leakage in negatively-charged membranes. Furthermore, confocal microscopy revealed that MW10 was located in the plasma membrane.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.1
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• pp.41-46
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• 2000

Using fluorescence polarization of 12-(9-anthroyloxy)stearic acid (12-AS) and 2-(9-anthroyloxy)stearic acid (2-AS), we evaluated the differential effects of local anesthetics on differential rotational rate between the surface (in carbon number 2 and its surroundings including the head group) and the hydrocarbon interior (in carbon number 12 and its surroundings) of the outer monolayer of the total lipid fraction liposome extracted from synaptosomal plasma membrane vesicles. The anisotropy (r) values for the hydrocarbon interior and the surface region of the liposome outer monolayer were $0.078{\pm}0.001$ and $0.114{\pm}0.001,$ respectively. This means that the rate of rotational mobility in the hydrocarbon interior is faster than that of the surface region. In a dose-dependent manner, the local anesthetics decreased the anisotropy of 12-AS in the hydrocarbon interior of the liposome outer monolayer but increased the anisotropy of 2-AS in the surface region of the monolayer. These results indicate that local anesthetics have significant disordering effects on the hydrocarbon interior but have significant ordering effects on the surface region of the liposome outer monolayer.

• Journal of Life Science
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• v.13 no.4
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• pp.463-472
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• 2003

Interactions between factor Va (HFVa) and membrane phosphatidylserine (PS) regulate the activity of the prothrombinase complex. I have previously shown that two solvent exposed hydrophobic residues located in the C2-domain, Trp2063 and Trp2064, are required for binding to immobilized PS and for expression of procoagulant activity on membranes containing 5% PS. In order to fully define the functional importance of these two residues I have expressed and isolated recombinant factor Va (rHFVa) W2063A/W2064A double mutant. In contrast to the native protein the two glycoforms resulting from alternative glycosylation of Asn2181 eluted as a single peak with rHFVa1 W2063A/W2064A eluting on the leading edge and rHFVa2 W2063A/W2064A eluting on the trailing edge. The double mutant rHFVa2 W2063A/W2064A expressed little or no procoagulant activity on membranes containing 1-10% mol % PS. In contrast, the procoagulant activity of this mutant was slightly greater than the native protein on membranes containing>18 mol % PS. The binding of rHFVa2 W2063A/W2064A to immobilized phospholipid vesicles was markedly reduced compared to the native protein in a surface plasmon resonance binding assay. I conclude that Trp2063 and Trp2064 are required for high affinity binding of factor Va to PS membranes and that this interaction is necessary for assembly of the prothrombinase complex on membranes containing physiological concentrations of PS.

• Applied Microscopy
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• v.29 no.3
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• pp.363-376
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• 1999

This study aims to demonstrate the effect of chitosan oligosaccharide on the ultrastructural changes in the mouse liver toxicated by carbon tetrachloride $(CCl_4)$. A healthy male ICR mouse that weighted $27{\pm}2gm$ was used for experiment. The experimental group was divided into three groups; the group A; the pretreated group with chitosan oligosaccharide, the group B; the simultaneous group, the group C; treated only the $CCl_4$. The group A was simultaneously treated with chitosan oligosaccharide and $CCl_4$ after pretreated with chitosan oligosaccharide for 7 days. The group B injected $CCl_4$ and chitosan oligosaccharide to the intraperitoneal. The group C injected with only $CCl_4$ to the intraperitoneal. The results were as follow: In the group A, the nuclear membrane and the mitochondria were observed almost normal in shapes at overall the time. Some lamellae of the RER (rough endoplasmic reticulum) destructed until 48 hours but ribosome attached. The destructed lamellae reformed at 72 hours but the smooth membrane vesicles not observed. The lysosomes observed at 72 hours. At 96 hours, all organelles showed in normal shapes. In the group B, changes of nuclear membranes were relatively lighter than group C. Mitochondria observed normal shape through the time. Parts of RER reformed the lamellae, other parts dilated inner cavity. And lipid droplet observed around the 24 hours. Glycogen and lysosome observed 48 hours and 72 hours, respectively. In the group C, nuclear membrane was irregular and nuclear cytoplasm condensed through the time. The lamellae of RER destructed from 24 to 96 hours. Smooth membrane vesicles observed in the cytoplasm at 48 ours. Mitochondria was less effected by toxic. And from the 24 hours, the variable sizes of lipid droplets observed in tile cytoplasm. These results suggest that chitosan oligosaccharide attenuates the toxic effect of the carbon tetrachloride in the mouse liver.