• 분석과학
• /
• 제26권1호
• /
• pp.106-112
• /
• 2013

Phosphodiesterase (PDE)는 세포내의 cAMP를 분해하는 효소로 세포의 신호 전달에 중요한 기능을 수행하는 것으로 알려져 왔다. 각각의 PDE들은 N-말단의 서열을 통해 세포 내 특정 부위로 이동되어 기능을 수행한다. 이전의 연구를 통해 바다달팽이인 군소에서 새롭게 클로닝된 ApPDE4 long-form이 원형질막과 시냅스전 뉴런의 말단에 발현됨을 확인하였다. 그러나, 현재까지 이러한 세포내 작용부위로의 이동, 즉 타겟팅(targeting)에 필요한 최소부위가 어디인지, 이러한 타겟팅이 세포에 미치는 영향은 무엇인지는 보고되지 않았다. 따라서, 본 연구에서는 이를 알아보기 위해 첫째, 원형질막으로 타겟팅에 필요한 최소부위를 알아 보고자 하였다. 이를 위해 다양한 결실돌연변이체를 제작하고, 이들의 이동과 분포를 확인한 결과, N-말단 13개의 아미노산만으로도 원형질막으로 타기팅에 충분하다는 것을 확인할 수 있었다. 또한, ApPDE4 N-말단의 20개 아미노산을 mRFP에 융합해서 만든 ApPDE4(N20)-mRFP를 HEK293T 세포에 과발현시킨 결과, 기포(bleb)가 생성되는 세포의 비정상적인 형태 변화가 관찰 되었다. 이러한 형태적 변화는 ApPDE4가 원형질막으로 타겟팅되는 것과 관련이 있었다. 대표적인 인지질의 하나인 PI4,$5P_2$에 선택적으로 결합함으로써 원형질막으로 타겟팅되는 단백질인 mRFP-$PLC{\delta}1$(PH)의 과발현도 ApPDE4(N20)-mRFP와 비슷한 세포의 형태적 변화가 유도됨을 확인할 수 있었다. ApPDE4의 N-말단은 PI4,$5P_2$와 같은 인지질과의 결합으로 원형질막으로 타겟팅될 수 있고, 형태적 변화를 유도하는 가능성을 제시한다.

• Journal of Periodontal and Implant Science
• /
• 제27권1호
• /
• pp.129-150
• /
• 1997

The purpose of this study was to evaluate the effects of tetracycline(TC}, flurbiprofen, and PDGF-BB loaded biodegradable membranes on the cell-attachment, the activity of loaded PDGF-BB, in vivo release kinetics, and guided bone regenerative potentials. To evaluate the cell attachment to membranes, the number of gingival fibroblasts attached to each membrane(10% TC, 10% flurbiprofen, $200ng/cm^2$ PDGF-BB loaded membranes, drug-unloaded membrane) was counted by coulter counter and the morphologic pattern of attached cells was examined under SEM. To determine whether the activity of loaded PDGF-BB is sustained, the cellular growth and survival rate of gingival fibroblasts was used for both standard PDGF-BB and loaded PDGF-BB. For evaluation of in vivo release kinetics, drug-loaded membranes were implanted on the dorsal skin of the rats. On 1, 3, 7, 10, 14, 21, and 28 days after implantation, the amount of remaining drugs were measured by HPLC assay for TC and flurbiprofen, and by ${\gamma}-scintillation$ counter for $PDGF-BB^{1125}$. For evaluation of guided regenerative potential, the amount of new bone in the calvarial defect(5mm in diameter) of the rat was measured by histomorphometry 1 and 2 weeks after implantation of membranes. The number of cells attached to the PDGF-BB loaded membrane was largest as compared with the other mernbranes.(p< 0.05) The activity of loaded PDGF-BB was not significantly different from the activity of standard PDGF-BB.(p<0.05) After initial burst release of drug during the first 24 hours, drugs were gradually released for 4 weeks. Especially the release rate of PDGF-BB was nearly constant during 4 weeks. PDGF-BB loaded membranes(200, $400ng/cm^2$) were effective in guided bone regeneration as compared with drug-unloaded membrane. These results implicate that drug-loaded biodegradable membranes might be a useful for guided bone regeneration.

• Journal of Microbiology and Biotechnology
• /
• 제18권2호
• /
• pp.248-254
• /
• 2008

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and two-dimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

• 멤브레인
• /
• 제15권1호
• /
• pp.70-83
• /
• 2005

폭기조를 간헐 폭기로 운전하여 호기/무산소(oxic/anoxic) 시간 비율과 무산소 조건에서 호기 조건으로의 전환시 공기세정이 투과플럭스 및 투과압력에 미치는 영향을 조사하였으며, 호기조건에서 무산소 조건으로 전환 시 펌프의 회전력에 의한 흡인압력이 투과플럭스와 투과 압력에 주는 영향을 조사하였다. 또한 긴 슬러지체류시간(SRT; Sludge Retention Time)과 고농도의 MLSS 유지시에 발생하는 미생물 대사산물이 분리막에 미치는 영향과 응집제를 투여하였을 때 분리막에 작용하는 플러스 효과에 대하여 조사하여 보았다. 호기/무산소(oxic/anoxic)의 시간비율에 따른 유기물 및 질소 제거효율을 조사해본 결과 폭기 40 비폭기 20분의 시간배분 조건에서 처리효율이 가장 양호하였으므로 호기/무산소(oxic/anoxic) 조건을 40/20분으로 한 조건(step-7)에서 약품주입 실험을 수행하였다. 액체 명반을 폭기조에 직접 투여할 경우 약품의 농도가 질산화 및 탈질 미생물에 많은 영향을 주는 것을 알 수 있었으며, MBR공정에서 인 1 mg/L를 제거하기 위해서는 약 0.7 mg/L의 액체명반이 필요한 것으로 확인되었다.

• Molecules and Cells
• /
• 제44권10호
• /
• pp.758-769
• /
• 2021

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I_CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I_CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I_CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I_CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

• BMB Reports
• /
• 제48권3호
• /
• pp.129-130
• /
• 2015

Autophagy is a tightly regulated lysosome-mediated catabolic process in eukaryotes that maintains cellular homeostasis. A distinguishable feature of autophagy is the formation of double- membrane structures, autophagosome, which envelopes the intracellular cargoes and finally degrades them by fusion with lysosomes. So far, many structures of Atg proteins working on the autophagosome formation have been reported, however those involved in autophagosome maturation, a fusion with lysosome, are relatively unknown. One of the molecules in autophagosome maturation, TECPR1, has been identified and recently, structural studies on both ATG5-TECPR1 and ATG5-ATG16L1 complexes revealed that TECPR1 and ATG16L1 share the same binding site on ATG5. These results, in combination with supporting biochemical and cellular biological data, provide an insight into a model for swapping ATG5 partners for autophagosome maturation.

• Biomolecules & Therapeutics
• /
• 제21권5호
• /
• pp.358-363
• /
• 2013

In the present study, we investigated the effect of intracellular glutathione (GSH) depletion in heart-derived H9c2 cells and its mechanism. L-buthionine-S,R-sulfoximine (BSO) induced the depletion of cellular GSH, and BSO-induced reactive oxygen species (ROS) production was inhibited by glutathione monoethyl ester (GME). Additionally, GME inhibited BSO-induced caspase-3 activation, annexin V-positive cells, and annexin V-negative/propidium iodide (PI)-positive cells. Treatment with rottlerin completely blocked BSO-induced cell death and ROS generation. BSO-induced GSH depletion caused a translocation of PKC-${\delta}$ from the cytosol to the membrane fraction, which was inhibited by treatment with GME. From these results, it is suggested that BSO-induced depletion of cellular GSH causes an activation of PKC-${\delta}$ and, subsequently, generation of ROS, thereby inducing H9c2 cell death.

• BMB Reports
• /
• 제35권1호
• /
• pp.24-27
• /
• 2002

Apoptotic cell death is an active process mediated by various signaling pathways, which include the caspase cascade and the stress-activated protein kinase pathways. The caspase cascade is activated by two distinct routes: one from cell surface and the other from mitochondria. Activation of the route from cell surface requires the cellular components that include membrane receptors, adaptor proteins such as TRADD and FADD, and caspase-8, while activation of the other from mitochondria requires Apaf-1, caspase-9, and cytosolic cytochrome c. On the other hand, persistent stimulation of the stress-activated protein kinase pathway is also shown to mediate apoptosis in many cell types. Gene-targeting studies with jnk- or jip-null mice, in particular, strongly suggest that this signaling pathway plays a pivotal role in the cellular machinery for apoptosis.

• Molecules and Cells
• /
• 제41권1호
• /
• pp.65-72
• /
• 2018

Autophagy is an evolutionally conserved cytoplasmic degradation system in which varieties of materials are sequestered by a double membrane structure, autophagosome, and delivered to the lysosomes for the degradation. Due to the wide varieties of targets, autophagic activity is essential for cellular homeostasis. Recent genetic evidence indicates that autophagy has a crucial role in the regulation of animal lifespan. Basal level of autophagic activity is elevated in many longevity paradigms and the activity is required for lifespan extension. In most cases, genes involved in autophagy and lysosomal function are induced by several transcription factors including HLH-30/TFEB, PHA-4/FOXA and MML-1/Mondo in long-lived animals. Pharmacological treatments have been shown to extend lifespan through activation of autophagy, indicating autophagy could be a potential and promising target to modulate animal lifespan. Here we summarize recent progress regarding the role of autophagy in lifespan regulation.

• 한국식품영양학회지
• /
• 제11권6호
• /
• pp.600-605
• /
• 1998

Present study was investigate to evaluate the aluminum absorption effect on lactic acid bacteria(Lactobacillus acidophilus ATTC 4356, Lactogacillus bulgaricus ATTC 11842, Lactobacillus casei IFO 3533, and Streptococcus thermophilus KCTC 2185 ; LAB) and Clostridium perfringens ATCC 3627 (CP) in artificial intestinal tract. Their growth rate, aluminum accumulation and cellular distribution was studied under anaerobic broth system. All of above microbes were inhibited by adding 10 to 100ppm of aluminum. The degree of aluminum in LAB (Lactobacillus acidophilus ATCC 4356, Lactobacillus bulgaricus ATCC 11842, Lactobacillus casei IFO 3533, and Streptococcus thermophilus KCTC 2185) was higher than of CP. The largest amount of aluminum was accumulated in Lactobacillus bulgaricus ATCC 11842. Aluminum accumulation in LAB was distributed in 49.1% at cell wall, 27.3% at plasma membrane, and 23.6% at cytoplasm, respectively. This study suggests that LAB might help to eliminate the ingested aluminum in intestinal tract.