The affiliation of the second author, Lei Sen Han, should be corrected.

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.
Binding Sites ; Calcium ; Calcium Signaling ; Calmodulin ; Ion Channels ; Ions ; Patch-Clamp Techniques ; Polycystic Kidney Diseases ; Transient Receptor Potential Channels

The present study was designed to examine the effect of heme oxygenase-1 (HO-1) induction by cobalt protoporphyrin (CoPP) on the cardiac functions and morphology, electrocardiogram (ECG) changes, myocardial antioxidants (superoxide dismutase [SOD] and glutathione [GSH]), and expression of heat shock protein (Hsp) 70 and connexin 43 (Cx-43) in myocardial muscles in isoproterenol (ISO) induced myocardial infarction (MI). Thirty two adult male Sprague Dawely rats were divided into 4 groups (each 8 rats): normal control (NC) group, ISO group: received ISO at dose of 150 mg/kg body weight intraperitoneally (i.p.) for 2 successive days; ISO + Trizma group: received (ISO) and Trizma (solvent of CoPP) at dose of 5 mg/kg i.p. injection 2 days before injection of ISO, with ISO at day 0 and at day 2 after ISO injections; and ISO + CoPP group: received ISO and CoPP at a dose of 5 mg/kg dissolved in Trizma i.p. injection as Trizma. We found that, administration of ISO caused significant increase in heart rate, corrected QT interval, ST segment, cardiac enzymes (lactate dehydrogenase, creatine kinase-muscle/brain), cardiac HO-1, Hsp70 with significant attenuation in myocardial GSH, SOD, and Cx-43. On the other hand, administration of CoPP caused significant improvement in ECG parameters, cardiac enzymes, cardiac morphology; antioxidants induced by ISO with significant increase in HO-1, Cx-43, and Hsp70 expression in myocardium. In conclusions, we concluded that induction of HO-1 by CoPP ameliorates ISO-induced myocardial injury, which might be due to up-regulation of Hsp70 and gap junction protein (Cx-43).
Adult ; Animals ; Antioxidants ; Body Weight ; Cobalt ; Connexin 43 ; Connexins ; Creatine ; Electrocardiography ; Glutathione ; Hand ; Heart Rate ; Heat-Shock Proteins ; Heme Oxygenase-1 ; Heme ; HSP70 Heat-Shock Proteins ; Humans ; Isoproterenol ; Male ; Muscles ; Myocardial Infarction ; Myocardium ; Oxidoreductases ; Rats ; Tromethamine ; Up-Regulation

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.
Calcium ; Carbachol ; Humans ; Hydrogen-Ion Concentration ; Ion Channels ; Kidney ; Mass Screening ; Mutagenesis, Site-Directed ; Mutant Proteins ; Protein Kinase C ; Receptors, Muscarinic

Curcumin, an active ingredient of Curcuma longa L., can reduce the concentration of low-density lipoproteins in plasma, in different ways. We had first reported that curcumin exhibits hypocholesterolemic properties by improving the apolipoprotein B (apoB) mRNA editing in primary rat hepatocytes. However, the role of curcumin in the regulation of apoB mRNA editing is not clear. Thus, we investigated the effect of curcumin on the expression of multiple editing components of apoB mRNA cytidine deamination to uridine (C-to-U) editosome. Our results demonstrated that treatment with 50 µM curcumin markedly increased the amount of edited apoB mRNA in primary mouse hepatocytes from 5.13%–8.05% to 27.63%–35.61%, and significantly elevated the levels of the core components apoB editing catalytic polypeptide-1 (APOBEC-1), apobec-1 complementation factor (ACF), and RNA-binding-motif-protein-47 (RBM47), as well as suppressed the level of the inhibitory component glycine-arginine-tyrosine-rich RNA binding protein. Moreover, the increased apoB RNA editing by 50 µM curcumin was significantly reduced by siRNA-mediated APOBEC-1, ACF, and RBM47 knockdown. These findings suggest that curcumin modulates apoB mRNA editing by coordinating the multiple editing components of the editosome in primary hepatocytes. Our data provided evidence for curcumin to be used therapeutically to prevent atherosclerosis.
Animals ; Apolipoproteins B ; Apolipoproteins ; Atherosclerosis ; Complement System Proteins ; Curcuma ; Curcumin ; Cytidine ; Deamination ; Hepatocytes ; Lipoproteins, LDL ; Mice ; Plasma ; Rats ; RNA Editing ; RNA, Messenger ; RNA-Binding Proteins ; Uridine

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of p27(Kip1), a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while p27(Kip1) expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of p27(Kip1) and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on p27(Kip1) expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and p27(Kip1)-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and p27(Kip1) expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and p27(Kip1) expression. In contrast, miR-186 expression positively associated with p27(Kip1) expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through p27(Kip1)-mediated cell cycle alternation.
Animals ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; Genes, Reporter ; Humans ; Luciferases ; Pituitary Neoplasms ; Rats ; Up-Regulation

Fumigaclavine C (FC), an active indole alkaloid, is obtained from endophytic Aspergillus terreus (strain No. FC118) by the root of Rhizophora stylosa (Rhizophoraceae). This study is designed to evaluate whether FC has anti-adipogenic effects in 3T3-L1 adipocytes and whether it ameliorates lipid accumulation in high-fat diet (HFD)-induced obese mice. FC notably increased the levels of glycerol in the culture supernatants and markedly reduced lipid accumulation in 3T3-L1 adipocytes. FC differentially inhibited the expressions of adipogenesis-related genes, including the peroxisome proliferator-activated receptor proteins, CCAAT/enhancer-binding proteins, and sterol regulatory element-binding proteins. FC markedly reduced the expressions of lipid synthesis-related genes, such as the fatty acid binding protein, lipoprotein lipase, and fatty acid synthase. Furthermore, FC significantly increased the expressions of lipolysis-related genes, such as the hormone-sensitive lipase, Aquaporin-7, and adipose triglyceride lipase. In HFD-induced obese mice, intraperitoneal injections of FC decreased both the body weight and visceral adipose tissue weight. FC administration significantly reduced lipid accumulation. Moreover, FC could dose-dependently and differentially regulate the expressions of lipid metabolism-related transcription factors. All these data indicated that FC exhibited anti-obesity effects through modulating adipogenesis and lipolysis.
Adipocytes ; Adipogenesis ; Animals ; Aspergillus ; Body Weight ; Carrier Proteins ; Diet, High-Fat ; Glycerol ; Injections, Intraperitoneal ; Intra-Abdominal Fat ; Lipase ; Lipolysis ; Lipoprotein Lipase ; Mice ; Mice, Obese ; Peroxisomes ; Rhizophoraceae ; Sterol Esterase ; Transcription Factors

Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.
Adult ; Animals ; Astrocytes ; Brain ; Embryonic Structures ; Epilepsy ; Epilepsy, Temporal Lobe ; Hippocampus ; Humans ; Immunohistochemistry ; Lymphangiogenesis ; Mice ; Microglia ; Pyramidal Cells ; Receptors, Vascular Endothelial Growth Factor ; Seizures ; Status Epilepticus ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor C ; Vascular Endothelial Growth Factor Receptor-3

The lamina II, also called the substantia gelatinosa (SG), of the trigeminal subnucleus caudalis (Vc), is thought to play an essential role in the control of orofacial nociception. Glycine and serotonin (5-hydroxytryptamine, 5-HT) are the important neurotransmitters that have the individual parts on the modulation of nociceptive transmission. However, the electrophysiological effects of 5-HT on the glycine receptors on SG neurons of the Vc have not been well studied yet. For this reason, we applied the whole-cell patch clamp technique to explore the interaction of intracellular signal transduction between 5-HT and the glycine receptors on SG neurons of the Vc in mice. In nine of 13 neurons tested (69.2%), pretreatment with 5-HT potentiated glycine-induced current (I(Gly)). Firstly, we examined with a 5-HT₁ receptor agonist (8-OH-DPAT, 5-HT(1/7) agonist, co-applied with SB-269970, 5-HT₇ antagonist) and antagonist (WAY-100635), but 5-HT₁ receptor agonist did not increase IGly and in the presence of 5-HT₁ antagonist, the potentiation of 5-HT on I(Gly) still happened. However, an agonist (α-methyl-5-HT) and antagonist (ketanserin) of the 5-HT₂ receptor mimicked and inhibited the enhancing effect of 5-HT on I(Gly) in the SG neurons, respectively. We also verified the role of the 5-HT₇ receptor by using a 5-HT₇ antagonist (SB-269970) but it also did not block the enhancement of 5-HT on I(Gly). Our study demonstrated that 5-HT facilitated I(Gly) in the SG neurons of the Vc through the 5-HT₂ receptor. The interaction between 5-HT and glycine appears to have a significant role in modulating the transmission of the nociceptive pathway.
Animals ; Glycine ; Mice ; Neurons ; Neurotransmitter Agents ; Nociception ; Patch-Clamp Techniques ; Receptors, Glycine ; Serotonin ; Signal Transduction ; Substantia Gelatinosa

Hydrogen sulfide is well-known to exhibit anti-inflammatory and cytoprotective activities, and also has protective effects in the liver. This study aimed to examine the protective effect of hydrogen sulfide in rats with hepatic encephalopathy, which was induced by mild bile duct ligation. In this rat model, bile ducts were mildly ligated for 26 days. Rats were treated for the final 5 days with sodium hydrosulfide (NaHS). NaHS (25 µmol/kg), 0.5% sodium carboxymethyl cellulose, or silymarin (100 mg/kg) was administered intraperitoneally once per day for 5 consecutive days. Mild bile duct ligation caused hepatotoxicity and inflammation in rats. Intraperitoneal NaHS administration reduced levels of aspartate aminotransferase and alanine aminotransferase, which are indicators of liver disease, compared to levels in the control mild bile duct ligation group. Levels of ammonia, a major causative factor of hepatic encephalopathy, were also significantly decreased. Malondialdehyde, myeloperoxidase, catalase, and tumor necrosis factor-α levels were measured to confirm antioxidative and anti-inflammatory effects. N-Methyl-D-aspartic acid (NMDA) receptors with neurotoxic activity were assessed for subunit NMDA receptor subtype 2B. Based on these data, NaHS is suggested to exhibit hepatoprotective effects and guard against neurotoxicity through antioxidant and anti-inflammatory actions.
Alanine Transaminase ; Ammonia ; Animals ; Aspartate Aminotransferases ; Bile Ducts ; Carboxymethylcellulose Sodium ; Catalase ; Hepatic Encephalopathy ; Hydrogen Sulfide ; Inflammation ; Ligation ; Liver ; Liver Diseases ; Malondialdehyde ; Models, Animal ; N-Methylaspartate ; Necrosis ; Peroxidase ; Rats ; Silymarin ; Sodium