• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.6
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• pp.539-547
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• 2019

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced $[Ca2^{+}]_i$ transient and reduced sarcoplasmic reticulum (SR) $Ca^{2+}$ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR $Ca^{2+}-ATPase$ subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises $Ca^{2+}$ signaling by downregulating the expression of DHPR and SERCA proteins.

• Journal of Life Science
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• v.19 no.8
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• pp.1055-1061
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• 2009

${\alpha}$-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are widespread throughout the central nervous system and appear to serve as synaptic receptors for fast excitatory synaptic transmission mediated by glutamate. Their modulation is believed to affect learning and memory. To identify the interaction proteins for the AMPA receptor subunit glutamate receptor-interacting protein 1 (GRIPl), GRIP1 interactions with armadillo family protein p0071/plakophilin-4 were investigated. GRIP1 protein bound to the tail region of p0071/plakophilin-4 but not to other armadillo family protein members in a yeast two-hybrid assay. The "S-X-V" motif at the carboxyl (C)-terminal end of p0071/plakophilin-4 is essential for interaction with GRIP1. p0071/plakophilin-4 interacted with the Postsynaptic density-95/Discs large/Zona occludens-1 (PDZ) domains of GRIPI in the yeast two-hybrid assay, as is indicated also by Glutathione S-transferase (GST) pull-down, and co-immunoprecipitated with GRIP1 antibody in brain fraction. The findings of this study provide evidence that p0071/plakophilin-4 is an interactor of GRIP1.

• YAKHAK HOEJI
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• v.39 no.6
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• pp.645-653
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• 1995

Dibenamine inhibited [$^{3}$H]quinuclidinyl benzilate ([$^{3}$H]QNB) binding in both concentration and incubation time-dependent manners. The $IC_{50}$/ value of dibenamine for the inhibition of the specific binding of 100 pM [$^{3}$'H]QNB following incubation of cerebral microsomes with dibenamine at 37.deg. C for 15 min was 20.mu.M. Dibenamine irreversibly decreased the binding site concentration for [$^{3}$H]QNB binding without affecting the affinity of [$^{3}$H]QNB for the muscarinic receptor. Analysis of the pirenzepine inhibition curve of [$^{3}$H]QNB binding to cerebral microsomes indicated the presence of two receptor subtypes with high(M$_{1}$ receptor, Ki=5nM) and low (M$_{2}$ receptor, Ki=160nM) affinity for pirenzepine. However, dibenamine(20.mu.M) treatment under the condition employed in these experiments caused steepening of the pirenzepine competition curve. The Ki value for pirenzepine in dibenamine treated-microsomes was approximately 120nM. suggesting a selective decrease in the number of M$_{1}$ receptor. Although dibenamine also inhibited [$^{3}$H]QNB binding to ventricular microsomes with $IC_{50}$/ value of 120.mu.M, the sensitivity for dibenamine in the ventricle was much lower than that in the cerebrum. These results indicate that dibenamine at low concentrations welectively inactivates the muscarinic M$_{1}$ receptor.

• Biomolecules & Therapeutics
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• v.9 no.3
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• pp.209-217
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• 2001

This study was attempted to investigate on renal effect of ($\pm$)6-chloro-7,8-dihydroxy-1-phenol 2,3,4,5-tetrahydro-lH-3 benzazepine (SKF 81297), dopamine $D_1$ receptor agonist, in dog. SKF 81297, when gluten intravenously, produced diuretic action along with the increases of renal plasma flow (RPF), glomerular filtration rate (GFR), amounts of N $a^{+}$ and $K^{+}$ excreted into urine ( $E_{Na}$ , $E_{K}$) and osmolar clearance ( $C_{osm}$). It also decreased the reabsorption rates of N $a^{+}$ and $K^{+}$ in renal tubule ( $R_{Na}$ , $R_{K}$) and free water clearance ( $C_{H2O}$), whereas ratios of $K^{+}$ agonist N $a^{+}$ in urine and filtration fraction (FF) was not changed. SKF 81297, when administered into a renal artery, elicited diuresis both in experimental kidney given the SKF 81297 and control kidney not given, while the effect was more remarkable in experimental kidney than those exhibited in control kidney. SKF 81297 given into carotid artery also exhibited diuresis, the potency at this time, compared to those induced by intravenous SKF 81297, was magnusgreat. Above results suggest that SKF 81297 produces diuresis by both indirect action through changes of central function and direct action being induced in kidney. Central diuretic action is mediated by improvement of renal hemodynamics, but direct action by inhibition of electrolytes reabsorption in renal tubule.enal tubule. tubule.

• Journal of Yeungnam Medical Science
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• v.11 no.2
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• pp.314-322
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• 1994

This study aimed to investigate the existence of GABA receptor and the mechanisms of action of GABA and diazepam on the trachealis muscle isolated from dog. Horizontal muscle strips of $2mm{\times}15mm$ were prepared from canine trachea, and isometric myography in isolated muscle chamber bubbled with 95/5%-$O_2/CO_2$at $36^{\circ}C$, at the pH of 7.4 was performed. Muscle strips contracted responding to the electrical field stimulation (ESP) by 2~20 Hz, 20 msec, monophasic square wave of 60 VDC GABA and diazepam suppressed the EFS-induced contractions to the similar extent, significantly. (p<0.05) Bicuculline, a $GABA_A$ receptor antagonist blocked both GABA- and diazepam- inhibitions; but DAVA, a $GABA_B$ receptor antagonist did not affect either of them. These results suggest that in the canine trachealis muscle, there may be only $GABA_A$ receptor, and GABA and diazepam inhibit the contractility via $GABA_A$A receptor.

• Archives of Pharmacal Research
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• v.12 no.4
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• pp.282-288
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• 1989

This study was investigated the effects of imipramine (IMI) and electroconvulsive shock (ECS), which are used as antidepressant therapy, on the central $\beta_1$or $\beta_2$ adrenergic receptor in anesthetized rats. The resting blood pressure and heart rate decreased in reserpinized group (5 mg/kg i. p., 24 hr before), but not in order 4 groups i. e. acute IMI (20 mg/kg i. p.. 3-5 hr before), chronic IMI (Same dose, twice a day for 14 days), siggle ECS (sinusoidal 20 Hz, 120 V for 2 sec) and repeated ECS (same condition, daily for 12 days). The increase of heart rate and hypotension evoked by 1 or 3 $\mu$g intracerebroventricular (i. c. v.) administration of (+) dobutamine, $\beta_2$-agonist, 1 or 3 $\mu$g i. c. v. was significantly attenuated in repeated ECS or reserpine treatment. And, the diminuation of pulse pressure of salbutamol also reduced by repeated ECS. These results suggest that IMI or ECS result in attenuation on tachycardia by (+) dobutamine or on hypotension by salbutamol, presumably by which the central $\beta_1$ or $\beta_2$receptor sensitivity may be suppressed, repectively.

• Journal of Acupuncture Research
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• v.21 no.6
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• pp.51-62
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• 2004

Objective: To investigate the analgesic effect and its serotonergic mechanism, especially related with 5-HTI and 5-HT2 receptor, of electroacupuncture(EA) in the rat model of collagen-induced arthritis(CIA). Methods : Immunization of male Sprague-Dawley rats with bovine type II (C II) collagen emulsified in Freund's incomplete adjuvant, followed by a booster injection 14 days later, leads to development of arthritis in more than 70% of rats by 21 days postinjection. After three weeks of first immunization, EA stimulation(2 Hz, 0.07 mA, 0.3 ms) was delivered into Jogsamni($ST_{36}$) for 30 minutes. Analgesic effect was evaluated by tail flick latency(TFL). We compared the analgesic effect of EA with TFLs between pretreatment of normal saline and pretreatment of spiroxatrine (5-HT1 receptor antagonist, 1mg/kg, intraperitoneal) and spiperone (5-HT2 receptor antagonist, 1 mg/kg, intraperitoneal) in CIA. Results : 1. TFLs were gradually decreased in CIA as increasing severity of arthritis. 2. Jogsamni($ST_{36}$) EA stimulation in CIA increased TFLs and the effect lasted for 60 minutes. 3. Increased TFLs with Jogsamni($ST_{36}$) EA stimulation were inhibited by pretreatment of spiroxatrine and spiperone in CIA. Conclusions : Jogsamni($ST_{36}$) EA showed analgesic effects in CIA The analgesic effects of Jogsamni($ST_{36}$) EA were inhibited by spiroxatrine and spiperone pretreatment. These observations suggest that 5-HT1 and 5-HT2 serotonergic receptor, which involve the release of serotonin neurotransmitter, play an important roles in analgesic mechanism of EA stimulation.

• Biomolecules & Therapeutics
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• v.3 no.4
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• pp.266-272
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. To generate and characterize a moloclonal antibody against $\beta$-adrenergic receptor, a synthetic $\beta$2-adrenergic receptor peptide (Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-lle-Asp-Val-Leu) which may comprise part of $\beta$-adrenergic receptor ligand binding pocket was coupled to Keyhole Limpet Hemocyanin (KLH) and used as an immunogen. Male BALB/C mice were immunized with this antigen and the immunized spleen was fused with myeloma SP2/0-Ag14 cells to produce monoclonal antibodies. Two clones were obtained but one of monoclonal antibodies, mAb5G09, was used throughout in this study because the other clone, mAb5All showed weak immunoreactivity against KLH as well. The mouse monoclonal antibody mAb5G09 produced in this study showed immunoreactivity to peptide-KLH conjugates and also to human A43l cells and guinea pig lung $\beta$2-adrenergic receptor as revealed by ELISA and western blot. In the course of determination of the effects of mAb5G09 on $\beta$-receptor ligand binding, it was observed that mAb5G09 specifically bound $\beta$-adrenergic radioligand [$^3$H]dihydroalprenolol (DHA) with a dissociation constant (Kd) of 60 nM. The [$^3$H]DHA binding activity of mAb5G09 had characteristics of immunoglobulins and the binding activity was not observed in the control anti-KLH monoclonal antibody. The monoclonal antibody, mAb5G09 produced in this study may provide useful models for the study of the structure of receptor binding sites.

• The Korean Journal of Pharmacology
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• v.32 no.2
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• pp.139-150
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• 1996

The effects of adenosine analogues on the electrically-evoked norepinephrine(NE) release and the influence of ischemia on the effects were studied in the rat hippocampus. Slices from the rat hippocampus were equilibrated with $0.1{\mu}M$ $[^3H]-norepinephrine$ and the release of the labelled product, $[^3H]-NE$, was evoked by electrical stimulation(3 Hz, 2 ms, 5 $VCm^{-1}$ and rectangular pulses for 90 sec), and the influence of various agents on the evoked tritium-outflow was investigated. Ischemia(15min with 95% $N_2$ +5% $CO_2$) increased both the basal and evoked NE release. These increases were abolished by addition of glucose into the superfused medium, and they were significantly inhibited either by $0.3\;{\mu}M$ tetrodotoxin pretreatment or by removing $Ca^{++}$ in the medium. MK-801$(1{sim}10\;{\mu}M)$, a specific NMDA receptor antagonist, and glibenclamide $(1\;{\mu}M)$, a $K^+-channel$ inhibitor, neither alter the evoked NE release nor affected the Ischemia-Induced increases in NE release. However, polymyxin B(0.03 mg), a specific protein kinase C inhibitor, inhibited the effect of ischemia on the evoked NE release. Adenosine and $N^6-cyclopentyladenosine$ decreased the NE release in a dose-dependent manner in ischemic condition, though the magnitude of inhibition was far less than those in normal (normoxic) condition. Also the treatment with $5{\mu}M$ DPCPX, a potent $A_1-adenosine$ receptor antagonist did not affect the ischemia-effect. These results suggest that the evoked-NE release is potentiated by ischemia, and this process being most probably mediated by protein kinase C, and that the decrease of NE release mediated through $A_1-adenosine$ receptor is significantly inhibited in ischemic state.