• Korean Chemical Engineering Research
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• 제58권2호
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• pp.280-285
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• 2020

We have developed a constitutive display system of the Pseudomonas fluorescens SIK W1 TliA lipase on the cell surface of Escherichia coli using E. coli outer membrane protein C (OmpC) as an anchoring motif, which is an economical compared to induced system. For the constitutive expression of truncated OmpC-TliA fusion proteins, gntT104 promoter was employed. Cell growth was not affected by over expression of fusion protein during entire culture time, suggesting cell lysis was not a problem. The localization of truncated OmpC-TliA fusion protein on the cell surface was confirmed by immunofluorescence microscopy and measuring whole cell lipase activity. Constitutively displayed lipase was very stable, retaining activity enantioselectivity throughout the five repeated reactions. These results suggest that OmpC from E. coli be a useful anchoring motif for displaying enzymes on the cell surface without any inducers, and this stable surface display system can be employed for a broad range of biotechnological applications.

• 대한수의학회지
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• 제46권4호
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• pp.355-361
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• 2006

Porcine epidemic diarrhea virus (PEDV) strain Chinju99, which was previously isolated from piglets suffering from severe diarrhea was used to characterize the membrane (M) protein gene to establish the molecular information, and the results will be useful in elucidating concepts related to molecular pathogenesis and antigenic structures of PEDV isolates. The Chinju99 M gene generated by reverse transcription and polymerase chain reaction (RT-PCR) consisted of 681 bases containing 22.3% adenine, 22.3% cytosine, 23.1% guanine and 32.3% thymine nucleotides, and the GC content was 45.4%. It had some nucleotide mismatches from M gene of other PEDV strains, such as CV777, Br1/87, KPEDV-9, JMe2, JS2004-2 and LJB-03 with 97-99% nucleotide sequence homology to these strains. Also, it encoded a protein of 226 amino acids, which had some mismatches from those of CV777, Br1/87, KPEDV-9, JMe2, JS20004-2 and LJB-03, as the amino acid sequence homology showed a 97-98% to these strains. The Chinju99 had a very close relationship to the Japanese strain JMe2 for the nucleotide and amino acid sequences of the M gene. The amino acids predicted from Chinju99 M gene consisted of mostly hydrophobic residues and contained three potential sites for asparagine (N)-linked glycosylation, two serine (S)-linked phosphorylation sites by protein kinase C, and two S- or threonine (T)-linked phosphorylation sites by casein kinase II.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.97-102
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• 1996

To investigate whether human $\beta$$_2$-adrenergic receptor devoid of the C-terminal two transmembrane helices retain its ligand binding activity and specificity, 5'780-bp DNA fragment of the receptor gene which encodes amino acid 1-260 of human $\beta$$_2$-adrenergic receptor was subcloned into the bacterial fusion protein expression vector and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was expressed as a membrane bound form which was verified by SDS-PAGE and Western blot. The fusion protein expressed in this study specifically bound $\beta$-adrenergic receptor ligand [$^3$H] Dihydroalprenolol. In saturation ligand binding assay, the $K_{d}$ value was 7.6 nM which was similar to that of intact $\beta$$_2$-adrenergic receptor in normal animal tissue ( $K_{d}$=1~2 nM) and the $B_{max}$ value was 266 fmol/mg membrane protein. In competition binding assay, the order of binding affinity of various adrenergic receptor agonists to the fusion protein was isoproterenol》epinephrine norepinephrine, which was similar to that of intact receptor in normal animal tissue. These results suggest that N-terminal five transmembrane helices of the $\beta$$_2$-adrenergic receptor be sufficient to determine the ligand binding activity and specificity, irrespective of the presence or absence of the C-terminal two transmembrane helices.s.s.s.

• Journal of Veterinary Science
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• 제21권2호
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• pp.33.1-33.15
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• 2020

Bovine ephemeral fever virus (BEFV) causes bovine ephemeral fever, which can produce considerable economic damage to the cattle industry. However, there is limited experimental evidence regarding the underlying mechanisms of BEFV. Annexin A2 (AnxA2) is a calcium and lipid-conjugated protein that binds phospholipids and the cytoskeleton in a Ca²⁺-dependent manner, and it participates in various cellular functions, including vesicular trafficking, organization of membrane domains, and virus proliferation. The role of the AnxA2 gene during virus infection has not yet been reported. In this study, we observed that AnxA2 gene expression was up-regulated in BHK-21 cells infected with the virus. Additionally, overexpression of the AnxA2 gene promoted the release of mature virus particles, whereas BEFV replication was remarkably inhibited after reducing AnxA2 gene expression by using the small interfering RNA (siRNA). For viral proteins, overexpression of the Matrix (M) gene promotes the release of mature virus particles. Moreover, the AnxA2 protein interaction with the M protein of BEFV was confirmed by GST pull-down and co-immunoprecipitation assays. Experimental results indicate that the C-terminal domain (268-334 aa) of AxnA2 contributes to this interaction. An additional mechanistic study showed that AnxA2 protein interacts with M protein and mediates the localization of the M protein at the plasma membrane. Furthermore, the absence of the AnxA2-V domain could attenuate the effect of AnxA2 on BEFV replication. These findings can contribute to elucidating the regulation of BEFV replication and may have implications for antiviral strategy development.

• 멤브레인
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• 제19권2호
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• pp.145-149
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• 2009

본 연구의 목적은 나노복합막을 제조하여 가수분해된 세리신 용액에 함유되어 있는 염의 제거 거동을 살펴보는 것이다. 계면중합법으로 나노복합막을 제조하고 상용화되어 있는 나노복합막과 탈염 거동을 비교하였다. 세리신 용액은 단백질 분해효소에 의해서 분해하여 저분자화하였고 전기영동에 의해 분자량 분포를 살펴보았다. 상대투과유량을 조사하여 나노복합막의 막오염 정도를 살펴보았다. 기공크기가 큰 나노복합막이 탈염에 효율적인 것을 확인하였다.

• Molecules and Cells
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• 제39권9호
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• pp.680-686
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• 2016

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that function to dissipate proton motive force and mitochondrial membrane potential. One UCP has been identified in Caenorhabditis elegans (C. elegans), namely UCP-4. In this study, we examined its expression and localization using a GFP marker in C. elegans. ucp-4 was expressed throughout the body from early embryo to aged adult and UCP-4 was localized in the mitochondria. It is known that increased mitochondrial membrane protential leads to a reactive oxygen species (ROS) increase, which is associated with age-related diseases, including neurodegenerative diseases in humans. A ucp-4 mutant showed increased mitochondrial membrane protential in association with increased neuronal defects during aging, and the neurons of ucp-4 overexpressing animals showed decreased neuronal defects during aging. These results suggest that UCP-4 may be involved in neuroprotection during aging via relieving mitochondrial membrane protential. We also investigated the relationship between UCP-4 and innate immunity because increased ROS can affect innate immunity. ucp-4 mutant displayed increased resistance to the pathogen Staphylococcus aureus compared to wild type. The enhanced immunity in the ucp-4 mutant could be related to increased mitochondrial membrane protential, presumably followed by increased ROS. In summary, UCP-4 might have an important role in neuronal aging and innate immune responses through mediating mitochondrial membrane protential.

• Applied Microscopy
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• 제1권1호
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• pp.11-17
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• 1969

Conidio spores of Aspergillus niger (strain No. NRRL 330) cultured on potato dextrose agar media were studied by electron microscopy, using the thin sectioning techniques. Conidio spores to be sectioned were fixed by triple methods with $K_2Cr_2O_7$, Glutaraldehyde and $OsO_4$. After dehydrated with alcohol, the specimens were embedded in metacrylate and epon resin media, and thinly sectioned by Porter-Blum MT-2. After sectioned these specimens were negative-stained with uranyl acetate and observed. by Hitachi HS-6 electron microscope. The results of this experiment were summarized as follows. 1. The structures of spore ,wall system seem to be formed 4 layers; exosporium, basal layer, spore coat and unit cell membrane. The protuberance of spore surface that was looked like hair appears to be protrusived from the basal layer. 2. The 3 layers of unit cell membrane was constituted outer layer membrane, inner layer membrane and inter-mediate light layer. 3. The structures of intra cytoplasmic membrane appear as spiral form which was consisted of 3 layers membrane system; outer membrane, inner membrane, and intermediate layer, which has pits. 4. The cement substance of spore coat and cortex may be changed quantitatively by physiological state in cell. 5. In some cases, we observed that the ribosome was transformed into poly ribosome group, and the storage materials and the protein crystals were changed variously. It. has been suggested that the morphological change of some cytoplasmic materials may be caused by some specialized function of the physiological stage.

• 한국막학회:학술대회논문집
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• 한국막학회 1998년도 춘계 총회 및 학술발표회
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• pp.97-98
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• 1998

1. Introduction : Polypropylene(PP) membrane is widely used in the field of microfiltration and ultrafiltration. However, the hydrophobicity of PP causes the adsorption of hydrophobic and amphoteric solutes in the feed. Surface modification techniques of membrane through the treatment of hydrophilizing agents, coating of hydrophilic compounds, UV, plasma and high energy irradiation, etc. can have a great effect on propensities to prevent the protein from staining membranes. Among them, the modification to hydophilize membrane surface using UV is very simple and effective. Recently many studies for more effective surface modification have been conducted. Iwata et al. prepared membranes by grafting polyethylene glycol diacrylate macromer(PEGDA) onto polysulfone with plasma using a glow discharge reactor which prevent the oil from staining the membrane. The primary mechanism contributing to the membranes is preventing the oil from directly contacting the surface of the membrane as the PEGDA chains dissolved into emulsion. To evaluate their feasibility for use as a anti-fouling separation membrane, we prepared hydrophilic membranes by UV irradiation method and investigated their characteristics.

• 한국환경보건학회지
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• 제19권4호
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• pp.83-89
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• 1993

To evaluate an effect of methanethiol on a cause of erythrocyte membrane damage in rats, methanethiol was given at 11.25 rag/100 g body weight, and after 4 hr, the animals were sacrifled, the activities of Na$^+$/K$^+$ ATPase, protein contents in partial purified erythrocyte membrane and erythrocyte indices were determined Concomitantly, in vitro, effect of methanethiol on the erythrocyte fragility, Na$^+$/K$^+$ ATPase activity and its kinetics in various concentration of substrate from the preincubated erythrocyte membrane with methanethiol were demonstrated. The spleen weight per body weight (%) and MCV of erythrocyte in methanethiol-treated rats were more increased than those in the control group. The Na$^+$/K$^+$ ATPase activities in erythrocyte membrane were more decreased in methanethiol-treated rats than those in the control group. The apply of 0.05 ng rat whole blood to the 0.24 mg/ng of methanethiol solution in isotonic condition showed the complete hemolysis. The Na$^+$/K$^+$ ATPase activity in preincubated erythrocyte membrane with methanethiol at 37$\circ$C showed the dual effect and the K$_m$ value of Na$^+$/K$^+$ ATPase was higher in the preincubated erythrocyte membrane with methanethiol than that in the preincubated erythrocyte membrane omitted the methanethiol. These results suggest that the methanethiol may induce the damage of rat's erythrocyte membrane due to a change in substrate binding affinity of Na$^+$/K$^+$ ATPase.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.37-45
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• 2022

The fungal cell wall and membrane are the principal targets of antifungals. Herein, we report that myricetin exerts antifungal activity against Candida albicans by damaging the cell wall integrity and notably enhancing the membrane permeability. In the presence of sorbitol, an osmotic protectant, the minimum inhibitory concentration (MIC) of myricetin against C. albicans increased from 20 to 40 and 80 ㎍/ml in 24 and 72 h, respectively, demonstrating that myricetin disturbs the cell wall integrity of C. albicans. Fluorescence microscopic images showed the presence of propidium iodide-stained C. albicans cells, indicating the myricetin-induced initial damage of the cell membrane. The effects of myricetin on the membrane permeability of C. albicans cells were assessed using crystal violet-uptake and intracellular material-leakage assays. The percentage uptakes of crystal violet for myricetin-treated C. albicans cells at 1×, 2×, and 4× the MIC of myricetin were 36.5, 60.6, and 79.4%, respectively, while those for DMSO-treated C. albicans cells were 28.2, 28.9, and 29.7%, respectively. Additionally, myricetin-treated C. albicans cells showed notable DNA and protein leakage, compared with the DMSO-treated controls. Furthermore, treatment of C. albicans cells with 1× the MIC of myricetin showed a 17.2 and 28.0% reduction in the binding of the lipophilic probes diphenylhexatriene and Nile red, respectively, indicating that myricetin alters the lipid components or order in the C. albicans cell membrane, leading to increased membrane permeability. Therefore, these data will provide insights into the pharmacological worth of myricetin as a prospective antifungal for treating C. albicans infections.