Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin- L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM Nlobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu- 593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin- L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM Nlobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu- 593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

The role of calmodulin in regulating calcium-permeable PKD2L1 channel activity

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.

Immunomodulating activity of Sargassum horneri extracts in RAW264.7 macrophages / 한국영양학회지

PURPOSE: Sargassum horneri (S. horneri) is a species of brown macroalgae that is common along the coast of Japan and Korea. The present study investigated the immuno-modulatory effects of different types of S. horneri extracts in RAW264.7 macrophages. METHODS: S. horneri was extracted by three different methods, hot water extraction, 50% ethanol extraction, and supercritical fluid extraction. Cell viability was then measured by MTT assay, while the production levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay and Griess assay, respectively. The expression and activation levels of inducible NO synthase (iNOS), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) were examined by western blot analysis. RESULTS: The three different S. horneri extracts were nontoxic against RAW 264.7 cells up to 50 µg/mL, among which treatment with hot water extract (HWE) of S. horneri significantly enhanced the production of TNF-α, IL-6, and NO in a dose-dependent manner. Hot water extract of S. horneri also increased the expression level of iNOS, suggesting that up-regulation of iNOS expression by HWE of S. horneri was responsible for the induction of NO production. In addition, treatment of RAW 264.7 macrophages with HWE of S. horneri increased the phosphorylation levels of ERK, p38 and JNK. Furthermore, the activation and subsequent nuclear translocation of NF-κB was enhanced upon treatment with HWE of S. horneri, indicating that HWE of S. horneri activates macrophages to secrete TNF-α, IL-6 and NO and induces iNOS expression via activation of the NF-κB and MAPKs signaling pathways. CONCLUSION: Taken together, these findings suggest that HWE of S. horneri possesses potential as a functional food with immunomodulatory activity.

Effects of Art & Culture Education on Behavior and Social Skill in Children: A Preliminary Study

OBJECTIVES: Art and culture education might play a good role in children's emotions ; however, few studies to verify this have been conducted. The objective of this study was to examine the effect of an art and culture education program on behavior and social skills in children aged 7-10 years. METHODS: Fifteen children (mean age 7.31+/-0.95 years) were recruited from a community center and participated in an art and culture education program. The participants received a once a week program for 17 sessions, and we compared scores on the Child Behavior Checklist (CBCL) and Social Skills Improvement System-Rating Scales (SSIS-RS) before and after intervention. RESULTS: The participants' subscale scores of CBCL-aggressive behavior, social immaturity, internalizing behavior, externalizing behavior, total behavior problems and social skill scores on the SSIS-RS showed improvement (p<.05) after 15-weeks of the art and culture education program. CONCLUSION: These results suggest a positive effect of an art and culture education program on the reduction of externalizing and internalizing behaviors and improvement of social skills in children. Further prospective, controlled studies in large samples are needed in order to confirm our findings.

Cloning of Mouse Pleckstrin 1 from Mast Cell Subtraction Library / 대한면역학회지

No abstract available.