• Molecules and Cells
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• 제37권4호
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• pp.307-313
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• 2014

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. Rap-GAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of migrating cells. Here, we examined localization of truncated RapGAP3 proteins and found that the I/LWEQ domain in the central region of RapGAP3 was sufficient for posterior localization in migrating cells, as opposed to leading-edge localization of full-length Rap-GAP3. All truncated proteins accumulated at the leading edge of migrating cells exhibited clear translocation to the cell cortex in response to stimulation, whereas proteins localized to the posterior in migrating cells displayed no translocation to the cortex. The I/LWEQ domain appears to passively accumulate at the posterior region in migrating cells due to exclusion from the extended front region in response to chemoattractant stimulation rather than actively being localized to the back of cells. Our results suggest that posterior localization of the I/LWEQ domain of RapGAP3 is likely related to F-actin, which has probably different properties compared to newly formed F-actin at the leading edge of migrating cells, at the lateral and posterior regions of the cell.

• Biomolecules & Therapeutics
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• 제25권3호
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• pp.272-278
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• 2017

Fucoidan has been reported to exhibit various beneficial activities ranging from to antivirus and anticancer properties. However, little information is available about the effects of fucoidan on cerebral ischemia-reperfusion injury (IRI). Our study aimed to explore the effects of fucoidan on cerebral IRI, as well as the underlying mechanisms. Sprague-Dawley (SD) rats were randomly subjected to four groups: Sham, IRI+saline (IRI+S), IRI+80 mg/kg fucoidan (IRI+F80), and IRI+160 mg/kg fucoidan (IRI+F160). Fucoidan (80 mg/kg or 160 mg/kg) was intraperitoneally injected from 7 days before the rats were induced to cerebral IRI model with middle cerebral artery occlusion (MCAO) method. At 24 h after reperfusion, neurological deficits and the total infarct volume were determined. The levels of inflammation-associated cytokines (interleukin (IL)-$1{\beta}$, IL-6, myeloperoxidase (MPO), and tumor necrosis factor (TNF)-${\alpha}$), oxidative stress-related proteins (malondialdehyde (MDA) and superoxide dismutase (SOD)) in the ischemic brain were measured by enzyme-linked immunosorbent assay (ELISA). Besides, the levels of apoptosis-related proteins (p-53, Bax, and B-cell lymphoma (Bcl)-2) and mitogen-activated protein kinase (MAPK) pathway (phosphorylation-extracellular signal-regulated kinase (p-ERK), p-c-Jun N-terminal kinase (JNK), and p-p38) were measured. Results showed that administration of fucoidan significantly reduced the neurological deficits and infarct volume compared to the IRI+S group in a dose-dependent manner. Also, fucoidan statistically decreased the levels of inflammation-associated cytokines, and oxidative stress-related proteins, inhibited apoptosis, and suppressed the MAPK pathway. So, Fucoidan plays a protective role in cerebral IRI might be by inhibition of MAPK pathway.

• Molecules and Cells
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• 제42권7호
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• pp.503-511
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• 2019

As sessile organisms, plants have developed sophisticated system to defend themselves against microbial attack. Since plants do not have specialized immune cells, all plant cells appear to have the innate ability to recognize pathogens and turn on an appropriate defense response. The plant innate immune system has two major branches: PAMPs (pathogen associated molecular patterns)-triggered immunity (PTI) and effector-triggered immunity (ETI). The ability to discriminate between self and non-self is a fundamental feature of living organisms, and it is a prerequisite for the activation of plant defenses specific to microbial infection. Arabidopsis cells express receptors that detect extracellular molecules or structures of the microbes, which are called collectively PAMPs and activate PTI. However, nucleotidebinding site leucine-rich repeats (NB-LRR) proteins mediated ETI is induced by direct or indirect recognition of effector molecules encoded by avr genes. In Arabidopsis, plasmamembrane localized multifunctional protein RIN4 (RPM1-interacting protein 4) plays important role in both PTI and ETI. Previous studies have suggested that RIN4 functions as a negative regulator of PTI. In addition, many different bacterial effector proteins modify RIN4 to destabilize plant immunity and several NB-LRR proteins, including RPM1 (resistance to Pseudomonas syringae pv. maculicola 1), RPS2 (resistance to P. syringae 2) guard RIN4. This review summarizes the current studies that have described signaling mechanism of RIN4 function, modification of RIN4 by bacterial effectors and different interacting partner of RIN4 in defense related pathway. In addition, the emerging role of the RIN4 in plant physiology and intercellular signaling as it presents in exosomes will be discussed.

• Journal of Microbiology and Biotechnology
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• 제34권3호
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• pp.525-537
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• 2024

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

• 생명과학회지
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• 제17권2호통권82호
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• pp.272-278
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• 2007

단백질분해효소는 다른 단백질들의 아미노산 간에 존재하는 peptide 결합을 절단하며 생리학적, 상업적 측면에서 중요한 위치를 차지하는 효소의 한 부류이다. 이러한 단백질분해효소의 새로운 공급원을 찾기 위하여 비교적 저온에서 체외단백질분해효소를 생산하는 세균을 동해심층수로부터 분리하였다. 분리된 균 중 저온에서의 생육정도와 높은 활성을 가지는 균주를 선별하여 HJ 47이라 명명하였다. 형태학적, 생리생화학적 특성과 16s rRNA gene의 염기서열을 조사한 후 Pseudoalteromonas sp.에 포함하는 것으로 나타났다. 분리된 Pseudoalteromonas sp. HJ 47은 $10^{\circ}C$에서도 비교적 잘 자랐으며, $37^{\circ}C$에서 최적의 생육을 보여 주었다. 최적생육온도와는 달리 배양시간당 최대 체외단백질분해효소의 생산은 $20^{\circ}C$에서 최대였고 대수기 후반과 정지기에 생산이 시작되어 15시간 경과 후 최대의 생산을 보여주었다. 효소활성의 최적온도는 $35^{\circ}C$, 최적 pH는 8로 판명되었다.

• Journal of Nutrition and Health
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• 제55권2호
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• pp.240-249
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• 2022

본 연구에서는 인간 유방암 세포 라인인 MDA-MB-231 세포에서 GD에 의해 EVs분비 억제에 의한 항암효과를 처음으로 확인하고자 하였다. MDA-MB-231 세포에 GD를 처리하였을 때 농도 의존적으로 세포의 증식률을 억제하는 것을 MTT assay를 통해 확인할 수 있었으며, ROS 염색과 apoptosis marker 단백질인 p-JNK단백질의 증가를 통해 GD에 의한 세포의 증식 억제 효과가 세포사멸에 의한 것임을 유추할 수 있었다. 또한 wound-healing assay, 세포 침윤 및 VEGF 농도를 측정한 결과 GD가 암세포의 이동, 전이 능력을 억제하는 것을 확인하였다. Nanosight를 통해서 MDA-MB-231 세포에서 분비되는 대조군 EVs 및 GD에 의해 변화된 EVs의 사이즈를 확인하였다. 마지막으로 GD를 처리한 MDA-MB-231 세포에서 분비된 EVs보다 GD를 처리하지 않은 대조군에서 분비된 EVs의 단백질 및 particles수가 유의적으로 감소하는 것을 확인을 하였다. 그리고 GD가 MDA-MB-231 세포에서 EVs분비를 감소시키는 것을 대표적인 exosome marker인 TSG101, CD63의 발현 감소로 확인할 수 있었다. 이러한 결과로 인해 GD가 암세포의 EVs 분비를 감소시켜 암세포의 성장 및 전이를 억제하였음을 확인하였다. 본 연구는 GD가 인간 유방암 세포인 MDA-MB-231 세포의 EVs 분비를 억제하는 효과가 있음을 제시하고 있다. 따라서 GD가 유방암의 화학요법 약물로 작용할 수 있음을 시사한다.

• Journal of Microbiology
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• 제41권3호
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• pp.175-182
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• 2003

Production of ribosomally synthesized antimicrobial peptides usually referred to as bacteriocins is an inducible trait in several gram positive bacteria, particularly in those belonging to the group of lactic acid bacteria. In many of these organisms, production of bacteriocins is inducible and induction requires secretion and extracellular accumulation of peptides that act as chemical messengers and trigger bacteriocin production. These inducer peptides are often referred to as autoinducers and are believed to permit a quorum sensing-based regulation of bacteriocin production. Notably, the peptides acting as autoinducers are dedicated peptides with or without antimicrobial activity or the bacteriocins themselves. The autoinducer-dependent induction of bacteriocin production requires histidine protein kinases and response regulator proteins of two-component signal transduction systems. The current working model for the regulation of class II bacteriocin production in lactic acid bacteria and the most relevant direct and indirect pieces of evidence supporting the model are discussed in this minireview.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.162-165
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• 2003

In order to study the secretion of the human urokinase-type plasminogen activator, u-PA, from the yeast yarrowia lipolytica, three kinds of integrative expression vector were constructed. These vectors differed only in their secretion control legions, pre-, pre-dip-(dipeptide Stretch) or pre-dip-pro sequences of the alkaline extracellular protease, which were joined inflame to the human u-PA cDNA. The recombinant Y. lipolytica Strains, transformed with the expression vectors, secreted the hyperglycosylated u-PA. A fibrin plate assay of the culture supernatants showed that the hyperglycosylated u-PA proteins could catalyze fibrinolysis, and that the pre-dip sequence was the most efficient secretory signal for the secretion of the u-PA from Y. lipolyica. This result suggests that Y. lipolytica can be developed as a potential host for the production of recombinant human u-PA.

• Animal cells and systems
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• 제13권3호
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• pp.283-288
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• 2009

The ubiquitous plasma membrane $Na^+/H^+$ exchanger 1 (NHE1) is rapidly activated in response to various extracellular stimuli and maintains normal cytoplasmic pH. Yeast two-hybrid screening was used in order to identify proteins interacting with NHE1 using its cytoplasmic domain as a bait from HeLa cDNA library. One of the interacting cDNA clones was human Lactate dehydrogenase B (LDHB). In vitro translated LDHB was pulled down together with GST-NHE1.cd protein in the GST pull down assay, confirming the interaction in vitro. LDHB antibody immunoprecipitated endogenous LDHB together with NHE1 from H9c2 cells, validating cellular interaction between NHE1 and LDHB. Subsequent analysis revealed that the overexpression of LDHB increased intracellular PH, implying opening of the NHE1 transporter. Moreover, overexpression of LDHB activated caspase 3 and induced cell death, consistent with the expected phenotype of hyper-activation of NHE1. Collectively, our data indicate that LDHB modulates NHE1 activity via physical interaction.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.753-758
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• 2000

Human leptin is identified as a 16kDa (146 amino acids) protein secreted from adipocytes which influences body weight homeostasis. In order to produce active leptin, the human obese gene coding for leptin was expressed in Bacillus subtilis WB600 strain which is deficient in six extracellular proteases. The recombinant leptin was produced in a culture supernatant, and in a culture supernatant, it was contained as high as 48% for total proteins. After simple purification steps, which consisted of ammonium sulfate precipitation and anion-exchange column chromatography, 2.3 mg of leptin with a purity greater than 95% was obtained from the 0.51 culture with the recovery yield of 38.3%. The purified leptin showed the correct folding structure with one disulfied bond.