• Molecules and Cells
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• 제37권7호
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• pp.526-531
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• 2014

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 Nterminal fragment encompassing NLS in PARP-$1^{+/+}$ and PARP-$1^{-/-}$ mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-$1^{+/+}$ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-$1^{-/-}$ cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.

• Molecules and Cells
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• 제40권9호
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• pp.643-654
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• 2017

Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the cis-Golgi localized t-SNARE protein Sed5 plays a role in endoplasmic reticulum (ER)-Golgi and intra-Golgi vesicular transport. We report that during autophagy, sed5-1 mutant cells could not properly transport Atg8 to the PAS, resulting in multiple Atg8 dots being dispersed into the cytoplasm. Some dots were trapped in the Golgi apparatus. Sed5 regulates the anterograde trafficking of Atg9-containing vesicles to the PAS by participating in the localization of Atg23 and Atg27 to the Golgi apparatus. Furthermore, we found that overexpression of SFT1 or SFT2 (suppressor of sed5 ts) rescued the autophagy defects in sed5-1 mutant cells. Our data suggest that Sed5 plays a novel role in autophagy, by regulating the formation of Atg9-containing vesicles in the Golgi apparatus, and the genetic interaction between Sft1/2 and Sed5 is essential for autophagy.

• Journal of Plant Biology
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• 제35권2호
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• pp.143-153
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• 1992

한국산 왕벚나무(Prunus yedoensis Matsumura) 화외밀선의 미세구조 및 당액 분비기작을 규명하기 위하여 광학현미경, 주사형 및 투과형 전자현미경을 이용하여 관찰하였다. 밀선은 secretory layer와 subsecretory layer로 구성되어 있었으며 엽병의 유관속은 subsecretory layer에 있는 유관속과 연결되어 있었다. Secretory cell에서는 cristae가 잘 발달되지 않은 다수의 mitochonidria가 관찰되었으나 소포체는 소수 나타났고 골지체가 전혀 관찰되지 않았다. 색소체는 thylakoid가 거의 발달되지 않았으며, 소수가 관찰되었고 $0.2-0.3\;\mu\textrm{m}$ 크기의 소포를 특징적으로 갖고 있었다. subsecretory cell에서는 원형질 연락사가 잘 발달해 있었고 다수의 Calcium oxalate 결정이 관찰되었는데 이는 당투과에 간접적으로 관여하는 것 같다. 당액 전구물질은 사부에서 subsecretory layer를 거쳐 secretory layer까지 이동하는 것을 원형질 연락사를 통한 symplastic transport에 의하여 일어나는 것으로 생각되며 secretory layer에서 당액으로 재합성되어 외부로 배출되는 것 같다. Secertory cell의 맨 바깥쪽 세포벽은 cuticle 에 의하여 덮여 있었으며 secretory cell에서 당액은 원형질막, 세포벽, cuticle을 차례로 통과하는 누출상 분비(eccrine secretion)에 의하여 분비되는 것으로 사료된다.

• Biomolecules & Therapeutics
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• 제16권1호
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• pp.14-20
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• 2008

In the present study, we examined how the transport of choline is regulated at the blood-brain barrier (BBB) under the central nervous system (CNS) cellular damages by oxidative stress using a conditionally immortalized rat brain capillary endothelial cells (TR-BBB), in vitro the BBB model. It was also tested whether the choline uptake is influenced by membrane potential, extracellular pH, protonophore (FCCP) and amiloride in TR-BBB cells. In result, $[^3H]choline$ uptake was inhibited by FCCP and dependent on extracellular pH. The treatment of TR-BBB cells with 20 ng/mL tumor necrosis $factor-{\alpha}$ $(TNF-{\alpha})$, 10 ng/mL lipopolysaccharide (LPS), 100 ${\mu}M$ diethyl maleate (DEM) and 100 ${\mu}M$ glutamate resulted in 3.0-fold, 2.6-fold, 1.8-fold and 2.0-fold increases of $[^3H]choline$ uptake at the respective peak time, respectively. In contrast, hydrogen peroxide and raffinose did not show any significant effects on choline uptake. In addition, choline efflux was significantly inhibited by $TNF-{\alpha}$, LPS and DEM producing cell damage states. In conclusion, the influx and efflux transport system for choline existed in TR-BBB cell line and this process was affected by several oxidative stress inducing agents.

• Environmental Analysis Health and Toxicology
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• 제20권1호
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• pp.87-95
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• 2005

Cadmium is well known as a toxic metal and has insulin mimicking effects in rat adipose tissue. This study was undertaken to investigate the effect of CdCl₂ on glucose transport and its mechanism in 3T3 - L1 adipocytes. CdCl₂ exhibits respectively 2.2 and 2.8 fold increases in the 2-deoxyglucose uptake when exposed to 10 and 25 μM of CdCl₂ for 12 hr. To investigate the stimulating mechanism of glucose transport induced by CdCl₂. Wortmannin and PD98059 were used respectively as PI3K inhibitor and MAPK inhibitor, which did not affect 2-DOG uptake. This results suggest that induced 2-deoxy-(l-3H)-D-glucose (2-DOG) uptake by CdCl₂ may not be concerned with the insulin signalling pathway. Whereas nifedipine, a calcium channel blocker inhibited the 2- DOG uptake stimulated by CdCl₂. In addition, we also measured the increased production of Reactive oxygen substances (ROS) and glutathione (GSH) level in 3T3-L1 adipocytes to investigate correlation between the glucose uptake and increased production of ROS with H2DCFDA. CdCl₂ increased production of ROS. Induced 2-DOG uptake and increased production of ROS by CdCl₂ were decreased by N-acetylcystein (NAC). And L-buthionine sulfoximine (BSO) a potent inhibitor of γ-GCS, decreased of 2-DOG uptake. Also NAC and BSO changed the cellular GSH level, but GSH/GSSG ratio remained unchanged at 10, 25 μM of CdCl₂.

• Journal of Pharmaceutical Investigation
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• 제34권6호
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• pp.483-489
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• 2004

Fexofenadine HCl is non-sedating histamine H1 receptor antagonist that can be used for the treatment of seasonal allergic rhinitis. The objective of this study was to investigate whether the carriers of deformable liposomes can enhance the transepithelial permeability of fexofenadine HCl across the in vitro ALI human nasal monolayer model. Characterization of this model was achieved by bioelectric measurements and morphological studies. The passage 2 and 3 of cell monolayers exhibited the TEER value of $2852\;{\pm}\;482\;ohm\;{\times}\;cm^2$ on 11 days of seeding and maintained high TEER value for 5 days. The deformable liposome of fexofenadine HCl was prepared with phosphatidylcholine (PC) and cholic acid using extruder method. The mean particle size was about 200 nm and the maximum entrapment efficiency of 33.0% was obtained in the formulation of 1% PC and $100\;{\mu}g/ml$ fexofenadine HCl. The toxicity of the deformable liposome to human nasal monolayers was evaluated by MTT assay and TEER value change. MTT assay showed that it has no toxic effect on the nasal epithelial cells in 2-hour incubation when the PC concentration was below 1%. However, deformable liposome could not enhance the transepithelial permeability $(P_{app})$ and cellular uptake of fexofenadine HCl. In conclusion, the in vitro model could be used in nasal drug transport studies and evaluation of transepithelial permeability of formulations.

• BMB Reports
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• 제54권7호
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• pp.380-385
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• 2021

Proper targeting of the βPAK-interacting exchange factor (βPIX)/G protein-coupled receptor kinase-interacting target protein (GIT) complex into distinct cellular compartments is essential for its diverse functions including neurite extension and synaptogenesis. However, the mechanism for translocation of this complex is still unknown. In the present study, we reported that the conventional kinesin, called kinesin-1, can transport the βPIX/GIT complex. Additionally, βPIX bind to KIF5A, a neuronal isoform of kinesin-1 heavy chain, but not KIF1 and KIF3. Mapping analysis revealed that the tail of KIF5s and LZ domain of βPIX were the respective binding domains. Silencing KIF5A or the expression of a variety of mutant forms of KIF5A inhibited βPIX targeting the neurite tips in PC12 cells. Furthermore, truncated mutants of βPIX without LZ domain did not interact with KIF5A, and were unable to target the neurite tips in PC12 cells. These results defined kinesin-1 as a motor protein of βPIX, and may provide new insights into βPIX/GIT complex-dependent neuronal pathophysiology.

• 한국해양환경ㆍ에너지학회지
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• 제16권1호
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• pp.9-16
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• 2013

해수 중 용존 이산화탄소 농도 증가가 두토막눈썹참갯지렁이(Perinereis aibuhitensis)의 세포내 에너지 할당(CEA, cellular energy allocation)에 미치는 영향을 알아보고자 0.39(대조구=390 ppmv), 3.03 (=3,030 ppmv), 10.3 (=10,300 ppmv), 그리고 30.1 (=30,100 ppmv) mM의 이산화탄소 농도를 갖는 해수에 청충을 7일간 노출하여 세포 에너지와 세포 전자전달체계(ETS, electro transport system) 활성을 분석하였다. 실험생물의 지질, 당질, 그리고 단백질 함량과 ETS로부터 에너지 소비율을 계산하여 CEA를 산출한 결과 이산화탄소 농도가 증가함에 따라 CEA가 감소하였다. 지질의 경우 이산화탄소 농도가 증가 할수록 지질함량도 증가하였으며, 당질은 이산화탄소 농도가 3.03 mM인 실험구에서 가장 낮고 10.3 mM인 실험구에서 일부 증가하였으나 가장 높은 농도에서는 다시 감소하는 경향을 보였다. 단백질은 이산화탄소 농도가 3.03 mM인 실험구에서는 영향이 없었지만, 10.3 mM 이상의 농도에서부터 유의한 수준의 CEA 감소가 나타났다. 에너지 소비율은 이산화탄소 최고 농도에서만 유의한 증가 현상이 나타났지만, CEA는 대조구와 비교해 3.03 mM의 실험구부터 감소하였다. 이산화탄소는 해수중 pH를 낮추어 시험생물에 스트레스를 증가시키고, 세포내 에너지 할당변화에 영향을 미친 것으로 판단된다. 해산 갯지렁이를 이용한 CEA 평가결과는 대기 중 이산화탄소의 증가 또는 이산화탄소 저감을 위해 추진되고 있는 해양 지중저장사업 과정에서 누출된 이산화탄소의 해양생태계 위해성을 예측하는 자료로 이용될 수 있을 것으로 판단된다.

• Molecules and Cells
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• 제27권6호
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• pp.615-620
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• 2009

The plant shoot meristem maintains a group of stem cells that remain active throughout the plant life. They continuously generate new cells that are then recruited for organ initiation in the peripheral zone. Stem cell proliferation and daughter cell differentiation has to be integrated with overall growth and development of the diverse functional domains within the shoot apex. Several studies have revealed extensive communication between these domains. The signaling mechanisms employed comprise diffusible peptides, directional transport of plant hormones, but also complex interactions between transcription factors, that together establish a panoply of regulatory inputs that fine-tune stem cell behavior in the shoot meristem.

• Journal of Applied Biological Chemistry
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• 제50권2호
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• pp.58-62
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• 2007

Identification of Dual-specificity protein phosphatase (DSP) substrates is essential in revealing physiological roles of DSPs. We isolated VHZ-interacting proteins from extracts of 293T cells overexpressing a VHZ (C95S, D65A) mutant known to be substrate- trapping mutant. Analysis of specific proteins bound to VHZ by 2D gel electrophoresis and mass spectroscopy revealed that these proteins contained Chaperonin containing TCP1, Type II phosphatidylinositol phosphate kinase ${\gamma}$, Intraflagellar transport 80 homolog, and Kinesin superfamily protein 1B. VHZ-interacting proteins showed that VHZ is involved in many important cellular signal pathways such as protein folding, molecular transportation, and tumor suppression.