• Molecules and Cells
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• v.43 no.11
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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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• v.37 no.sup2
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• pp.447-463
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• 2007

Periodontal ligament (PDL) cells have been known as multipotential cells, and as playing an important rolesin periodontal regeneration. The PDL cells are composed of heterogeneous cell populations which have the capacity to differentiate into either cementoblasts or osteoblasts, depending on needs and conditions. Therefore, PDL cells have the capacity to produce mineralized nodules in vitro in mineralization medium which include ascorbic acid, ${\beta}$-glycerophosphate and dexamethasone. In spite of these well-known osteoblast like properties of PDL cells, very little is known about the molecules involved in the formation of the mineralized nodules in the PDL cells. In the present study, we analysed gene-expression profiles during the mineralization process of cultured PDL cells by means of a cDNA microarray consisting of 3063 genes. Nodules of mineralized matrix were strongly stained with alizarin red S on the PDL cells cultured in the media with mineralization supplements. Among 3,063 genes analyzed, 35 were up-regulated more than two-fold at one or more time points in cells that developed matrix mineralization nodules, and 38 were down-regulated to less than half their normal level of expression. In accord with the morphological change we observed, several genes related to calcium-related or mineral metabolism were induced in PDL cells during osteogenesis, such as IGF-II and IGFBP-2. Proteogycan 1, fibulin-5, keratin 5, ,${\beta}$-actin, ${\alpha}$-smooth muscle actin and capping protein, and cytoskeleton and extracellular matrix proteins were up-regulated during mineralization. Several genes encoding proteins related to apoptosis weredifferentially expressed in PDL cells cultured in the medium containing mineralization supplements. Dkk-I and Nip3, which are apoptosis-inducing agents, were up-regulated, and Btf and TAXlBP1, which have an anti-apoptosis activity, were down-regulated during mineralization. Also periostin and S100 calciumbinding protein A4 were down-regulated during mineralization.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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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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• v.24 no.4
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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.