OBJECTIVES: To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism. METHODS: FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-In^TM T-REx^TM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation. RESULTS: After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine. CONCLUSIONS This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.
Calcium-Binding Proteins/metabolism* ; Cell Line ; Humans ; Myocardium/metabolism* ; Phosphorylation

Ingestion of food affects secretion of hormones from enteroendocrine cells located in the gastrointestinal mucosa. These hormones are involved in the regulation of various gastrointestinal functions including the control of food intake. One cell in the stomach, the X/A-like has received much attention over the past years due to the production of ghrelin. Until now, ghrelin is the only known orexigenic hormone that is peripherally produced and centrally acting to stimulate food intake. Subsequently, additional peptide products of this cell have been described including desacyl ghrelin, obestatin and nesfatin-1. Desacyl ghrelin seems to be involved in the regulation of food intake as well and could play a counter-balancing role of ghrelin's orexigenic effect. In contrast, the initially proposed anorexigenic action of obestatin did not hold true and therefore the involvement of this peptide in the regulation of feeding is questionable. Lastly, the identification of nesfatin-1 in the same cell in different vesicles than ghrelin extended the function of this cell type to the inhibition of feeding. Therefore, this X/A-like cell could play a unique role by encompassing yin and yang properties to mediate not only hunger but also satiety.
Calcium-Binding Proteins ; DNA-Binding Proteins ; Eating ; Enteroendocrine Cells ; Ghrelin ; Hunger ; Mucous Membrane ; Nerve Tissue Proteins ; Obesity ; Stomach

Humans ; Retroperitoneal Neoplasms ; Liposarcoma ; Calcium-Binding Proteins ; Mixed Function Oxygenases ; Membrane Proteins ; Muscle Proteins

The distributions of calretinin (CR)- and parvalbumin (PV)-immunoreactive neurons in the main olfactory bulb (MOB) of the goat were examined in this study. As in other animals, the goat MOB has a characteristic laminar structure with laminar types and distribution patterns in each layer. CR-immunoreaction was observed in all layers of the MOB, except for the olfactory nerve layer. Most of CR-immunoreactive neurons were observed in the glomerular and granule cell layers. Relatively small number of CR-immunoreactive neurons was detected in other layers. These CR-immunoreactive neurons were interneurons. PV-immunoreaction was detected in all layers. In contrast to CR, olfactory nerve bundles were immunostained with PV. Most of PV-immunoreactive neurons were distributed in the glomerular and granule cell layers. PV-immunoreactive neurons were interneurons. This result suggests that CR and PV may play important roles in the olfactory signal modulation through interneurons in the goat MOB.
Animals ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Goats ; Immunohistochemistry ; Interneurons ; Neurons ; Olfactory Bulb ; Olfactory Nerve ; Smell

Calbindin D-28K (CALB) is one of the calcium-binding proteins which is assumed to be buffering, transport of Ca2+, and regulation of various enzyme systems. In the spinal cord, a subpopulation of calbindin-immunoreactive neurons located in the ventral portion of lamina VII, medial to the motoneuron column, has recently been proposed to be Renshaw cells (RCs), that mediate recurrent inhibition of spinal alpha-motoneurons, based on the anatomical location. In this study, we have performed to investigate the correlation between RCs containing high levels of CALB and motoneurons in the ventral horn of lumbar spinal cord of the ataxic pogo mice, that characterized by a failures of interlimb coordination, and prolonged excessive tone of hindlimb extensor muscles. We have shown that CALB immunoreactive RCs was significantly decreased in the ventral horn of lumbar spinal cord of the ataxic pogo mice (p.0.05), when compared with the control mice. Whereas, CALB immunoreactivity expression levels were no difference in the dorsal horn. Furthermore, CALB protein was significantly decreased in the lumbar spinal cord of the ataxic pogo mice (p.0.01). However, there were no difference in the cervical and thoracic spinal cord of the between control and pogo mice. These results suggest that motoneurons of ventral horn of the lumbar spinal cord might be more excited state, results in the decreased CALB immunoreactive RCs have not mediated a motoneuron excitability, in the atxic mice, pogo.
Animals ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Hindlimb ; Horns ; Mice ; Muscles ; Neurons ; Spinal Cord

Lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, has been reported to be an intercellular signaling molecule. LPE mobilizes intracellular Ca²⁺ through G-protein-coupled receptor (GPCR) in some cells types. However, GPCRs for lysophosphatidic acid (LPA) were not implicated in the LPE-mediated activities in LPA GPCR overexpression systems or in SK-OV3 ovarian cancer cells. In the present study, in human SH-SY5Y neuroblastoma cells, experiments with LPA₁ antagonists showed LPE induced intracellular Ca²⁺ increases in an LPA₁ GPCR-dependent manner. Furthermore, LPE increased intracellular Ca²⁺ through pertussis-sensitive G proteins, edelfosine-sensitive-phospholipase C, 2-APB-sensitive IP₃ receptors, Ca²⁺ release from intracellular Ca²⁺ stores, and subsequent Ca²⁺ influx across plasma membranes, and LPA acted on LPA₁ and LPA₂ receptors to induce Ca²⁺ response in a 2-APB-sensitive and insensitive manner. These findings suggest novel involvements for LPE and LPA in calcium signaling in human SH-SY5Y neuroblastoma cells.
Calcium Signaling* ; Calcium* ; Cell Membrane ; GTP-Binding Proteins ; Humans* ; Neuroblastoma* ; Ovarian Neoplasms

Recent human genetic studies have shown that Gβ5 is related to various clinical symptoms, such as sinus bradycardia, cognitive disability, and attention deficit hyperactivity disorder. Although the calcium signaling cascade is closely associated with a heterotrimeric G-protein, the function of Gβ5 in calcium signaling and its relevance to clinical symptoms remain unknown. In this study, we investigated the in vitro changes of store-operated calcium entry (SOCE) with exogenous expression of Gβ5. The cells expressing Gβ5 had enhanced SOCE after depletion of calcium ion inside the endoplasmic reticulum. Gβ5 also augmented Stim1- and Orai1-dependent SOCE. An ORAI1 loss-of-function mutant did not show inhibition of Gβ5-induced SOCE, and a STIM1-ERM truncation mutant showed no enhancement of SOCE. These results suggested a novel role of GNB5 and Stim1, and provided insight into the regulatory mechanism of SOCE.
Attention Deficit Disorder with Hyperactivity ; Bradycardia ; Calcium ; Calcium Signaling ; Endoplasmic Reticulum ; GTP-Binding Proteins ; Humans ; In Vitro Techniques

Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca2+ levels. Ca2+ binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS-/- mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca2+ levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca2+ homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca2+ homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.
Animals ; Calcium ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Carrier Proteins ; Cerebral Cortex ; Homeostasis ; Immunohistochemistry ; Mice ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase ; Synaptic Transmission

The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past two decades. Store-operated Ca2+-release-activated Ca2+ (CRAC) channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement. In a set of breakthrough studies over the past two years, stromal interaction molecule 1 (STIM1, the ER Ca2+ sensor) and Orai1 (a pore-forming subunit of the CRAC channel) have been identified. Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels (SOCCs).
Animals ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Calcium Signaling ; drug effects ; Humans ; Membrane Proteins ; antagonists & inhibitors ; metabolism ; Protein Binding

OBJECTIVETo observe the expression of new myoepithelial cell markers calponin and P63 in human submandibular glands.

METHODSCalponin and P63 antigen in routinely processed human submandibular gland tissues were immunohistochemically demonstrated by monoclonal antibodies to calponin and P63.

RESULTSCalponin expressed around all acinus and intercalated ducts as linear or punctuate pattern. Positive staining was also noted in peripheral area of some thin striated ducts that connect to intercalated ducts. Subulate or trigonal calponin expression was sometimes seen between the duct dells of striated ducts. P63 expressed mainly in the nucleus of the basal cells of excretory duct.

CONCLUSIONCalponin is an ideal gland. P63 labels mainly the basal cells of excretory duct. marker for myoepithelial cells of human submandibular