• Molecules and Cells
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• 제37권4호
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• pp.322-329
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• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the ${\gamma}$-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

• Journal of Ginseng Research
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• 제43권2호
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• pp.326-334
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• 2019

Background: The objective of our study was to analyze the neuroprotective effects of ginsenoside derivatives Rb1, Rb2, Rc, Rd, Rg1, and Rg3 against glutamate-mediated neurotoxicity in HT22 hippocampal mouse neuron cells. Methods: The neuroprotective effect of ginsenosides were evaluated by measuring cell viability. Protein expressions of mitogen-activated protein kinase (MAPK), Bcl2, Bax, and apoptosis-inducing factor (AIF) were determined by Western blot analysis. The occurrence of apoptotic and death cells was determined by flow cytometry. Cellular level of $Ca^{2+}$ and reactive oxygen species (ROS) levels were evaluated by image analysis using the fluorescent probes Fluor-3 and 2',7'-dichlorodihydrofluorescein diacetate, respectively. In vivo efficacy of neuroprotection was evaluated using the Mongolian gerbil of ischemic brain injury model. Result: Reduction of cell viability by glutamate (5 mM) was significantly suppressed by treatment with ginsenoside Rb2. Phosphorylation of MAPKs, Bax, and nuclear AIF was gradually increased by treatment with 5 mM of glutamate and decreased by co-treatment with Rb2. The occurrence of apoptotic cells was decreased by treatment with Rb2 ($25.7{\mu}M$). Cellular $Ca^{2+}$ and ROS levels were decreased in the presence of Rb2, and in vivo data indicated that Rb2 treatment (10 mg/kg) significantly diminished the number of degenerated neurons. Conclusion: Our results suggest that Rb2 possesses neuroprotective properties that suppress glutamate-induced neurotoxicity. The molecular mechanism of Rb2 is by suppressing the MAPKs activity and AIF translocation.

• 생명과학회지
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• 제17권2호통권82호
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• pp.198-203
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• 2007

형질전환은 유전자의 기능을 이해하는데 매우 중요한 기법이다. $Ca^{2+}$-인산 침전법은 시간과 비용이 저렴하여 가장 흔히 사용된다. 그러나 성숙 신경세포는 어린 신경세포나 다른 세포종에 비하여 형질전환이 어렵고 쉽게 죽는다. 본 연구에서는 Clontech사의 $CalPhos^{TM}$ Mammalian Transfection 방법을 수정하여 성숙한 신경세포를 효율적으로 형질전환할 수 있는 방법을 고안하였다. 대뇌 신경교세포를 DMEM/10% 말혈청에서 70-80% confluence까지 키우고 배지를 혈청이 첨가되지 않은 Neurobasal/Ara-C로 바꾸어 주어 더 이상 신경교세포가 분열하지 않게 한 다음, 여기에 E19 해마신경세포를 접종하여 배양하였다. $DNA/Ca^{2+}$-인산 침전물은 Clontech사의 $CalPhos^{TM}$ Mammalian Transfection Kit을 이용하여 크기($0.5-1\;{\mu}m$ in diameter) 및 농도(약 10 particles/$100\;{\mu}m^2$)를 배지에서 배양시간을 변화시켜 적당히 조절하였다. 이렇게 하면 in vitro에서 2주 이상 배양한 신경세포도 24-well plate 한 well당 10-15개의 형질전환된 건강한 신경세포를 얻을 수 있었다. 이 방법의 효용성을 검증하기 위하여 연접단백질인 $EGFP-CaMKII{\alpha}$ 융합단백질과 RFP 단백질 유전자(각각 $pEGFP-CaMKII{\alpha}$ 및 pDsRed2)를 형질전환한 결과 전자는 점박이 모양, 후자는 세포전체에 퍼진 양상의 표현을 관찰할 수 있었다. 따라서 본 연구는 성숙한 신경세포를 효율적으로 형질전환할 수 있는 방법을 제공한다.

• 생명과학회지
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• 제30권11호
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• pp.939-946
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• 2020

뇌졸중은 전 세계적으로 신경계 장애를 일으키는 주요 원인 중 하나이며, 뇌졸중 환자는 다양한 운동, 인지 및 정신 장애를 나타낸다. GPR88은 orphan G protein coupled receptor이며 striatal medium spiny neurons에서 높게 발현이 되며, GPR88이 결손이 된 경우 motor coordination과 motor learning에 문제가 발생하게 된다. 본 연구에서는 Western blot 및 real-time PCR을 사용하여 허혈성 마우스 모델에서 GPR88 발현이 감소함을 발견 하였다. 또한, 뇌에서 유래한 세 가지 유형의 세포들, 뇌혈관내피세포(brain microvascular endothelial cells), 미세 아교세포(microglial cells) 및 신경 세포들에서 GPR88의 발현정도를 확인한 결과, HT22 신경 세포에서 GPR88의 발현이 가장 높음을 관찰하였고, 뇌졸중과 유사한 실험조건인 oxygen glucose deprivation (OGD) 조건에 배양한 HT22 신경세포에서 GPR88의 발현이 감소하였다. 또한 GPR88 효현제인 RTI-13951-33 (10 mg/kg)을 전처리후에 뇌허혈을 유발하였을 때, infarct volume의 감소, vestibular-motor function 및 neurological score의 개선효과를 관찰할 수 있었다. 이러한 결과는 GPR88이 허혈성 뇌졸중을 포함한 CNS 질환의 치료를 위한 잠재적인 약물표적이 될 수 있음을 제시한다.

• 생명과학회지
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• 제19권1호
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• pp.21-33
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• 2009

대황(Rhei Rhizoma; RR, 대황(大黃))은 Rheum officinale Baill.와 Rheum palmatum L.(polygonaceae)의 땅속부분으로 남아시아의 민속의학에서 간 및 신장의 손상을 치료하는데 널리 이용되고 있다. 본 연구에서는 배양한 흰쥐 해마신경세포의 저산소증모델을 이용하여 대황의 물추출물이 유전자 표현에 미치는 영향을 microarray 방법을 이용하여 조사하였다. 배양 후 10일 (DIV10)에 추출물을 배지에 $2.5{\mu}g/ml$ 농도로 첨가하고, DIV13에 저산소증(2% $O_2$/5% $CO_2$, $37^{\circ}C$, 3 h)을 유발한 후 24 시간 후에 total RNA를 분리하여 microarray에 사용하였다. MA-plot에 의하면 표현이 연화된 대부분의 유전자는 ${\pm}2$배 이내로 증감되었다. 이 가운데 Global M 값이 0.2(즉, 15%)보다 더 증가한 유전자는 472종, Global M 값이 -0.2(즉, -15%)보다 더 감소한 유전자는 725종이였다. 세포의 생존과 관련된 유전자 가운데 세포자연사 억제유전자인 Tegt(2.4배), Nfkb1 (2.4배), Veg (1.8배), Ngfr (1.6배) 등이 크게 증가하였으며, 반면에 자연사 촉진유전자인 Bad (-64%), Cstb (-66%)는 감소하였다. 스트레스를 극복하는데 필요한 유전자인 Defb3 (2.7배), Cygb (2.2배), Ahsg (2.18배), Alox5 (2배) 등도 크게 증가하였다. 그리고 세포 성장을 촉진하는 유전자인 Erbb2 (1.84배), Mapk12 (1.8배)도 크게 증가하였다. 따라서 대황의 물추출물은 세포생존에 필요한 유전자를 증가시키고, 세포사를 유도하는 유전자는 감소시킴으로서 저산소층 스트레스에서 신경세포의 사망을 억제하는 것으로 해석된다.

• 사상체질의학회지
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• 제27권2호
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• pp.270-287
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• 2015

Objectives To evaluate the neuroprotective effects of modified Yuldahanso-tang (MYH) in a Parkinson's disease mouse model. Methods 1) Four groups (each of 8 rats per group) were used in this study. 2) The neuroprotective effect of MYH was examined in a Parkinson's disease mouse model. C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day), intraperitoneal (i.p.) for 5 days. 3) The brains of 2 mice per group were removed and frozen at $-20^{\circ}C$, and the striatum-substantia nigra part was seperated. The protein volume was measured by Bradford method following Bio-Rad protein analyzing kit. Using mouse/Rat Dopamine ELISA Assay Kit. 4) The brains of 2 mice per group were separated and removed. TH-immunohistochemical was examined in the MPTP-induced Parkinson's disease mice to evaluate the neuroprotective effects of MYH on ST and SNpc. 5) Two mice out of each group were anesthetized and skulls were opened from occipital to frontal direction to take out the brains. The brains added TTC solution for 20 minutes for staining. 6) The water tank used for morris water maze test was filled with $28^{\circ}C$ water, and a round platform of 10cm in diameter was installed for mice to step on. The study was carried out once a day within 30 seconds, keep exercising to step on the platform in the pool. 7) The brains of two mice out of each group were fixed in 10% formaldehyde solution and paraphillin substance was infiltrated. They were fragmented by microtome, and observed under an optical microscope after Hematoxylin & Eosin staining. 8) A round acrylic cylinder with its upper side open was filled with clean water and depressive mouse models were forced to swim for 15 minutes. After 24 hours the animals were put in the same equipment for 5 minutes and were forced to swim. 9) The convenient, simple, and accurate high-performance liquid chromatography (HPLC) method was established for simultaneous determination of Neurotransmitters in MPTP-MYH group. Results 1) MYH possess Dopamine cell protective effect on MPTP-induced injury in striatum and substantia nigra pars compacta. 2) MYH inhibits the loss of tyrosine hydroxylase-immunoreacitive (TH-IR) cells in the striatum and substantia nigra pars compacta on MPTP-induced injury in C57BL/6 mice. 3) MYH possesses improvement effect on MPTP-induced memory deterioration in C57BL/6 mice through the reduction of prolongated Sort of lost time by MPTP injection using the Morris water maze test. 4) MYH possesses hippocampal neuron protective effect on MPTP-induced injury in C57BL/6 mice. 5) MYH possesses improvement effect on MPTP-induced motor behaviour deficits and depression in C57BL/6 mice through the reduction of prolongated losing motion by MPTP injection using the Forced swimming test. 6) MYH increases serotonin product amount on MPTP-induced injury in C57BL/6 mice. Conclusions This experiment suggests that the neuroprotective effect of MYH is mediated by the increase in Dopamin, TH-ir cell, Hippocampus and Serotonin. Furthermore, MYH essential oil may serve as a potential preventive or therapeutic agent regarding Parkinson's disease.