• Molecules and Cells
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• v.19 no.3
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• pp.356-364
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• 2005

Intramolecular excimer formation of 1,3-di(1-pyrenyl) propane(Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of lateral and rotational mobilities of bulk bilayer structures of synaptosomal plasma membrane vesicles (SPMVs) from the bovine cerebral cortex. Ethanol increased the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py in the SPMVs. Selective quenching of both DPH and Py-3-Py by trinitrophenyl groups was used to examine the range of transbilayer asymmetric rotational mobility and the rate and range of transbilayer asymmetric lateral mobility of SPMVs. Ethanol increased the rotational and lateral mobility of the outer monolayer more than of the inner one. Thus ethanol has a selective fluidizing effect within the transbilayer domains of the SPMVs. Radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py was used to examine both the effect of ethanol on annular lipid fluidity and protein distribution in the SPMVs. Ethanol increased annular lipid fluidity and also caused membrane proteins to cluster. These effects on neuronal membranes may be responsible for some, though not all, of the general anesthetic actions of ethanol.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.274-280
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• 2005

Pasteurella multocida is known to cause widespread infections in husbandry. To induce homologous and heterologous immunity against the infections, outer membrane proteins (OMPs) in the envelope of P. multocida are thought to be attractive vaccine candidates. Outer membrane protein H is considered as the major component of OMPs. In this study, a gene for OmpH was isolated from pathogenic P. multocida serogroup A. The gene was composed of 1,047 nucleotides coding 348 amino acids with signal peptide of 20 amino acids. The amino acid composition showed about 80 to 98 per cent sequence homologies among other 10 strains of P. multocida serogroup A, reported so far. A recombinant ompH, from which signal peptide was truncated, was generated using pRSET A to name 'pRSET A/OmpH-F2'. The pRSET A/OmpH-F2 was well expressed in E. coli BL21(DE3). The truncated OmpH was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Its molecular weight was registered to be 40 kDa on SDS-PAGE gel. In order to generate immunesera against the OmpH, 50 ug of the protein was intraperitoneally injected into mice three times. The anti-OmpH immuneserum recognized about $5{\times}10^{-2}$ng quantity of the purified OmpH. It can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (Serogroup A).

• Journal of Life Science
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• v.18 no.11
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• pp.1606-1611
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• 2008

Outer membrane proteins (OMPs) expressed in the Gram negative bacteria such as Salmonella play multiple functions including material transports, adhesive factors and reception of external signals. This study has been focused on an OmpW protein known as a protein required to form a hydrophobic porin in outer membrane. We have constructed a S. typhimurium CK10 mutant deleting an ompW gene on chromosome. The CK10 strain was more tolerant to SDS than the wild-type strain did. As increase of salt concentration in the culture media, significantly decreased amount of OmpW protein in cells were detected. The maximum OmpW protein was expressed in the absence of salt supplement. However, the growth of CK10 strain was indistinguishable compared to that of the wild-type strain at the variable osmotic conditions. The biological role of differential OmpW expression in response to osmotic conditions remains to be investigated.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.307-313
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• 2001

Whether there exists a sympathetic neural mechanism regulating the expression of aquaporin (AQP) water channels in the kidney was investigated. Male Sprague-Dawley rats were treated with reserpine (1 mg/kg, IP), and the expression of AQP1-4 proteins was determined in the kidney one day thereafter. Following the treatment with reserpine, the systolic blood pressure measured in a conscious state was significantly decreased in the experimental group compared with that in the control $(83{\pm}8\;vs\;124{\pm}6\;mmHg;\;n=6\;each,\;P＜0.05)$. The expression of AQP2 proteins was decreased in the cortex, outer medulla, and inner medulla. The decrease of AQP2 proteins was in parallel in the membrane and the cytoplasmic fractions, suggesting a preserved AQP2 targeting. No significant changes were observed in the expression of AQP1, AQP3, or AQP4. Neither basal nor AVP-stimulated formation of cAMP was significantly altered. These results suggest that the sympathetic nervous system has a tonic stimulatory effect specifically on the expression of AQP2 water channels in the kidney.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.141-146
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• 2005

A new system for displaying proteins on the surface of Escherichia coli was developed using the Salmonella typhimurium outer membrane protein C (OmpC) as an anchoring motif. The C-terminal deletionfusion strategy was developed to fuse the polyhistidine peptides and green fluorescent protein (GFP) to the Cterminal of the truncated functional portion of OmpC. The polyhistidine peptides of up to 243 amino acids could besuccessfully displayed on the E. coli cell surface, which allowed recombinant E. coli to adsorb up to 34.2 μmol of Cd2+ per gram dry cell weight. The GFP could also be successfully displayed on the E. coli cell surface. These results suggest that the C-terminal deletion-fusion strategy employing the S. typhimurium OmpC as an anchoring motif provides a new efficient way for the display of large proteins on the surface of E. coli.

• Molecules and Cells
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• v.27 no.3
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• pp.321-327
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• 2009

Voltage-dependent anion channels (VDACs) are reported to be porin-type, ${\beta}$-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.

• Korean Journal of Plant Tissue Culture
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• v.24 no.4
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• pp.197-201
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• 1997

Plant seed oil-bodies or oleosomes ate the repository of the neutral lipid stored in seeds. These organelles in many oilseeds may comprise half of the total cellular volume. Oleosomes are surrounded by a half-unit membrane of phospholipid into which are embedded proteins called oleosins. Oleosins are present at high density on the oil-body surface and after storage proteins comprise the most abundant proteins in oilseeds. Oleosins are specifically targeted and anchored to oil-bodies after co-translation on the ER. It has been shown that the amino-acid sequences responsible for this unique targeting reside primarily in the central hydrophobic tore of the oleosin polypeptide. In addition, a signal-like sequence is found near the junction of the hydrophobic domain and ann N-terminal hydrophilic / amphipathic domain. This "signal" which is uncleaved is also essential for correct targeting. Oil-bodies and their associated oleosins may be recovered by floatation centrifugation of aqueous seed extracts. This simple partitioning step results in a dramatic enrichment for oleosins in the oil-body fraction. In the light of these properties, we reasoned that it would be feasible to create fusion proteins on oil-bodies comprising oleosins and an additional valuable protein of pharmaceutical or industrial interest. It was further postulated that if these proteins were displayed on the outer surface of oil-bodies, it would be possible to release them from the purified oil-bodies using chemical or proteolytic cleavage. This could result in a simple means of recovering high-value protein from seeds at a significant (i.e. commercial) scale. This procedure has been successfully reduced to practice for a wide variety of proteins of therapeutic, industrial and food no. The utillity of the method will be discussed using a blood anticoagulant, hirudin, and industrial enzymes as key examples.

• Archives of Pharmacal Research
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• v.27 no.6
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• pp.653-661
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• 2004

Fluorescence probes located in different membrane regions were used to evaluate the effect of dopamine$.$HCI on the structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and thickness of the lipid bilayer) of synaptosomal plasma membrane vesicles (SPMV), which were obtained from the bovine cerebral cortex. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, and radiationless energy transfer from the tryptophan of membrane pro-teins to Py-3-Py and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS) was also utilized. Dopamine$.$HCI increased both the bulk lateral mobility and annular lipid fluidity, and it had a greater fluidizing effect on the inner monolayer than on the outer monolayer. Furthermore, the drug had a clustering effect on membrane proteins.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.1097-1103
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• 2020

Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V¹⁴⁰, V¹⁷⁶, K¹⁷⁹, V²²⁶, V²³², and K²³⁴, which were truncated from the C-terminal end of the mipA gene (MV¹⁴⁰, MV¹⁷⁶, MV¹⁷⁹, MV²²⁶, MV²³², and MV²³⁴) were examined. The whole-cell lipase activities showed that MV¹⁴⁰ was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV¹⁴⁰ as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV¹⁴⁰. Additionally the MV¹⁴⁰ motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV¹⁴⁰ motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.

• The Korean Journal of Nuclear Medicine
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• v.33 no.1
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• pp.1-10
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• 1999

Cancer cells are known to show increased rates of glycolysis metabolism. Based on this, PET studies using F-18-fluorodeoxyglucose have been used for the detection of primary and metastatic tumors. To account for this increased glucose uptake, a variety of mechanisms has been proposed. Glucose influx across the cell membrane is mediated by a family of structurally related proteins known as glucose transporters (Gluts). Among 6 isoforms of Gluts, Glut-1 and/or Glut-3 have been reported to show increased expression in various tumors. Increased level of Glut mRNA transcription is supposed to be the basic mechanism of Glut overexpression at the protein level. Some oncogens such as src or ras intensely stimulate Glut-1 by means of increased Glut-1 mRNA levels. Hexokinase activity is another important factor in glucose uptake in cancer cells. Especially hexokinase type II is considered to be involved in glycolysis of cancer cells. Much of the hexokinase of tumor cells is bound to outer membrane of mitochondria by the porin, a hexokinase receptor. Through this interaction, hexokinase may gain preferred access to ATP synthesized via oxidative phosphorylation in the inner mitochondria compartment. Other biologic factors such as tumor blood flow, blood volume, hypoxia, and infiltrating cells in tumor tissue are involved. Relative hypoxia may activate the anaerobic glycotytic pathway. Surrounding macrophages and newly formed granulation tissue in tumor showed greater glucose uptake than did viable cancer cells. To expand the application of FDG PET in oncology, it is important for nuclear medicine physicians to understand the related mechanisms of glucose uptake in cancer tissue.