• 생명과학회지
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• 제20권1호
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• pp.1-8
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• 2010

본 연구자들의 앞선 연구에서는 햄스터 상구에서 AMPA 수용체의 아형인 GluR1과 GluR4의 분포와 눈 적출 이후 이들 수용체의 분포를 면역세포화학적 방법으로 연구하였다. 또한, 표지한 GluR1과 GluR4를 칼슘결합단백질인 calbindin D28K, calretinin, parvalbumin과 GABA로 표지하여 비교하였다. 본 연구에서 역행성이동추적물질(retrograde tracer)인 호스래디시퍼옥시다아제(horseradish peroxidase, HRP)를 상구의 각 주요 상행로와 하행로에 주입함으로써 GluR1- 면역반응 신경세포들과 GluR4- 면역반응 신경세포들이 투사신경세포(projection neurons)임을 밝혀내었다. Tecto-reticulospinal pathway (TRS)와 dorsal lateral geniculate nucleus (dLGN)으로 HRP 를 주입한후, 햄스터들은 회복을 위해 48시간 동안 살려둔 뒤 관류(perfusion)하였다. GluR- 면역반응 처리된 절편들은 역행성 표지된 신경세포를 지님을 확인하였다. HRP를 주입하였더니 단지 적은 수의 GluR1- 면역반응 신경세포들이 TRS (1.4%)와 dLGN (2.6%)으로 투사되었고, 반면에 많은 수의 GluR4- 면역반응 신경세포들이 TRS (32.7%)로 투사되었다. 사이/투사 신경세포(inter/projection neurons)들은 GluR 아단위들의 분류된 분포와 차별화된 양상을 보였고 이들의 이러한 분포는 칼슘결합단백질들과 GABA와는 겹쳐지지 않았으며, 일전에 발표했던 시각적 행동 반응에서 안구 적출 후 수용체 아단위들의 기능적 다양화와는 차별화된 양상을 보였다.

• 생명과학회지
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• 제20권10호
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine (5-HT))는 신경계의 세포-세포 간의 신호전달의 주요한 신경전달물질이다. 세포막에 존재하는 serotonin transporter (SERT)는 연접간격에 존재하는 5-HT를 세포 내로 재흡수 하여 세포외부의 5-HT 농도를 조절하지만 그 기전은 아직 밝혀지지 않았다. 본 연구에서는 yeast two-hybrid system을 사용하여 SERT의 C-말단이 protein kinase C-$\varepsilon$ (PKC-$\varepsilon$)과 특이적으로 결합함을 알았다. PKC-$\varepsilon$는 PKC의 isotype으로 calcium 비의존적이며 phorbol ester/diacylglycerol 민감성 serine/threonine kinase이다. $Na^+/Cl^-$ 의존성 SLC6 gene family의 다른 수송체는 PKC-$\varepsilon$과 결합하지 않았다. Deletion mutant들을 사용하여 SERT는 PKC-$\varepsilon$의 C-말단부위와 결합함을 알았으며, 또한 이 단백질간의 결합을 GST pull-down assay로 확인하였다. PKC-$\varepsilon$는 in vitro에서 SERT의 N-말단의 펩티드를 인산화시켰다. 이러한 결과들은 PKC-$\varepsilon$에 의한 SERT의 인산화가 세포막에 존재하는 SERT의 활성을 조절하는 역할을 할 가능성을 시사한다.

• Molecules and Cells
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• 제43권11호
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• Journal of Periodontal and Implant Science
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• 제37권sup2호
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• pp.447-463
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• 2007

치주인대세포는 시험관적 실험에서 광물화 결절형성을 유도할 수 있으므로 광물화 결절형성에 관여하는 유전자들을 특이하게 발현할 것으로 여겨진다. 이에 본 실험은 cDNA microarray를 이용한 동시 유전자분석을 시행하여 치주인대세포의 분화에 의한 광물화 결절형성시 나타나는 유전자의 특징적 발현 양상을 알아보고자 하였다. 교정치료를 목적으로 경북대학교병원에 내원한 환자의 제일소구치를 발치하여 통상적 방법으로 치주인대세포를 분리, 배양하였고, 3세대의 치주인대세포를 사용하여 실험을 시행하였다. 치주인대세포를 100mm 배양접시에 넣고 배양하여 매 2일 마다 배지를 교환해 주고, 10% FBS만을 투여한 대조군으로, ascorbic acid $(50\;{\mu}g/ml)$, ${\beta}-glycerophosphate$ (10 mM) 및 100 nM dexamethasone을 투여한 군을 실험군으로 하였다. 배양된 치주인대세포에 ascorbic acid, ${\beta}-glycerophosphate$, 그리고 dexamethasone을 투여한 실험군에서 21일째 광물화된 결정을 관찰할 수 있었으나 대조군에서는 관찰할 수 없었다. 3063개의 유전자를 분석한 결과 35개 유전자가 대조군에 비해 2배이상 발현이 증가하였고, 38개 유전자는 2배이상 발현이 감소하였다. 형태학적 검사에서 보여준 바와 같이 광물화 형성과정시 관여하는 JGF-2과 IGFBP2와 같은 유전자가 실험군에서 증가하였으며, 세포골격과 세포외기질 형성에 관여하는 proteogycan 1, fibulin-5, keratin 5, ${\beta}-actin$, ${\alpha}-smooth$ muscle actin, capping protein 등도 발현이 실험군에서 증가하였다. 한편 periostin and S100 calcium-binding protein A4는 대조군에서 오히려 높게 나타나므로 이는 배양된 치주인대세포가 그 자체의 표현형을 유지하고 있음을 보여 주고 있다. 그 외 apoptosis를 유발시키는데 관여하는 Dkk-1와 Nip3는 실험군에서 높게 발현되었고, apoptosis를 억제시키는데 관여하는 Btf와 TAX1BP1는 오히려 낮게 발현됨을 알 수 있으므로 이는 실험군에서 치주인대세포가 골아세포로의 분화되었음을 나타낸다.

• The Korean Journal of Physiology and Pharmacology
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• 제17권3호
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

• Applied Microscopy
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• 제24권4호
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• pp.61-77
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• 1994

The calcium-binding proteins (CaBP), parvalbumin (PV) and calbindin-D 28K (calbindin) are particularly abundant and specific in their distribution, and present in different subsets of neurons in many brain regions. Although their physiological roles in the neurons have not been elucidated, they are valuable markers of neuronal subpopulations for anatomical and developmental studies. This study is designed to characterize dorsal lateral geniculate nucleus (dLGN) neurons and axon terminals in terms of differential expression of immunoreactivity (IR) for two well-known CaBPs, PV and calbindin. The experiments were carried out on 6 adult monkeys. Monkeys were perfused under deep Nembutal anesthesia with 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer. After removal, the brains were postfixed for 6-8 hr in 2% paraformaldehyde at $4^{\circ}C$ and infiltrated with 30% sucrose at $4^{\circ}C$. Thereafter, they were frozen in dry ice. Serial sections of the thalamus, at $20{\mu}m$, were made in the frontal plane with a sliding microtome. The sections were stained for PV and calbindin with indirect immunocytochemical methods. For electron microscopy, after infiltration with 30% sucrose the blocks of thalamus were serially sectioned at $50{\mu}m$ with a Vibratome in the coronal plane and stained immediately by indirect ABC methods without Triton X-100 in incubation medium. Stained sections were postfixed in 0.2% osmium tetroxide, dehydrated and flat-embedded in Spurr resin. The block was then trimmed to contain only a selected lamina or interlaminar space. The dLGN proper showed strong PV IR in fibers in all laminae and interlaminar zones. Particularly dense staining was noted in layers 1 and 2 that contain many stained fibers from optic tract. Neuronal cell body stained with PV was concentrated only in the laminae. In these laminae staining was moderate in cell bodies of all large and medium-sized neurons, and was strong in cell bodies of some small neurons together with their processes. Calbindin IR was marked in the neuronal cell body and neuropil in the S layers and interlaminar zones whereas moderate in the neuropil throughout the nucleus. Regional difference in distribution of PV and calbindin IR cell is distinct; the former is only in the laminae and the latter in both the S layer and interlaminar space. The CaBP-IR elements were confined to about $10{\mu}m$ in depth of Vibratome section. The IR product for CaBP was mainly associated with synaptic vesicle, pre- and post-synaptic membrane, and outer mitochondrial membrane and along microtubule. PV-IR was noted in various neuronal elements such as neuronal soma, dendrite, RLP, F, PSD and some myelinated or unmyelinated axons, and was not seen in the RSD and glial cells. Only a few neuronal components in dLGN was IR for calbindin and its reaction product was less dense than that of PV, and scattered throughout cytoplasm of soma of some relay neurons, and was also persent in some dendrite, myelinated axons and RLP. The RSD, F, PSD and glial elements were always non-IR for calbindin. Calbindin labelled RLP were presynaptic to unlabeled dendrite or dendritic spine and PSD. Calbindin-labeled dendrite of various sizes were always postsynaptic to unlabeled RSD, RLP or F. From this study it is suggested that dLGN cells of different functional systems and their differential projection to the visual cortex can be distinguished by differential expression of PV and calbindin.