Colchicine modulates calcium homeostasis and electrical property of HL‐1 cells

Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca²⁺) homeostasis and attenuate the electrical effects of the extracellular matrix on atrial myocytes. Whole‐cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL‐1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine‐treated HL‐1 cells had a longer action potential duration with smaller intracellular Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content by 10% and 47%, respectively. Colchicine‐treated HL‐1 cells showed a smaller L‐type Ca²⁺ current, reverse mode sodium–calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra‐rapid activating outward potassium current and apamin‐sensitive small‐conductance Ca²⁺‐activated potassium current compared with control cells. Colchicine‐treated HL‐1 cells expressed less SERCA2a, total, Thr17‐phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16‐phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen‐induced action potential duration in HL‐1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca²⁺ regulation and attenuates the electrical effects of collagen, which may contribute to its anti‐AF activity.     

Ca2+ Signaling and Cell Death Induced by Protriptyline in HepG2 Human Hepatoma Cells

The effect of protriptyline on Ca²⁺ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca²⁺]_i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50–150 μM) evoked [Ca²⁺]_i rises. The Ca²⁺ entry was inhibited by removal of Ca²⁺. Protriptyline‐induced Ca²⁺ entry was confirmed by Mn²⁺‐induced quench of fura‐2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12‐myristate 13 acetate did not inhibit Ca²⁺ entry. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 40% of protriptyline‐induced response. Treatment with protriptyline abolished BHQ‐induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline‐evoked response by 70%. At 20–40 μM, protriptyline killed cells which was not reversed by the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca²⁺]_i rises that involved Ca²⁺ entry through nifedipine‐sensitive Ca²⁺ channels and PLC‐dependent Ca²⁺ release from endoplasmic reticulum. Protriptyline induced Ca²⁺‐independent cell death.     

Magnolol induces the distributional changes of p160 and adipose differentiation-related protein in adrenal cells

Magnolol stimulates adrenal steroidogenesis and induces the distributional changes of p160 and adipose differentiation-related protein (ADRP) in rat adrenal cells. This study investigated the underlying signaling mechanisms involved in these processes. Magnolol (30 M) caused a time-dependent increase in the phosphorylation of extracellular signal-related kinase (ERK) in cultured adrenal cells. The following evidence supports a link between ERK activation and p160 translocation. First, the magnolol-induced redistribution of p160 from the lipid droplet surface to the cytosol, resulting in the decrease in the percentages of p160-positive cells, and this decrease in p160-positive cells was completely blocked by pretreatment with either of the MAPK-ERK kinase (MEK) inhibitors PD98059 or U0126. Second, magnolol did not significantly decrease total p160 protein levels but caused an increase in threonine phosphorylation of p160, which reached a maximum after 5 min of magnolol treatment, and this magnolol-induced phosphorylation of p160 was prevented by pretreatment with U0126, suggesting the involvement of ERK. In addition, magnolol decreased both ADRP immunostaining intensity at the lipid droplet surface and the percentage of ADRP-positive cells. This was further confirmed biochemically by the decrease in ADRP levels in total cell homogenates and in lipid droplet fractions. Magnolol-induced decrease in ADRP staining at the lipid droplet surface was not affected by pretreatment with PD98059 or U0126, indicating that ERK signaling was not involved in this event. Furthermore, treatment with 30 M magnolol for 6 h resulted in about 50% decrease in ADRP protein level. Therefore, decreased protein levels of p160 and ADRP at the lipid droplet surface induced by magnolol were mediated via two different mechanisms: phosphorylation of p160 and downregulation of ADRP expression, respectively.     

Critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease

The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and K(d) value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1-3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1-4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.     

Isolation and characterization of enantioselective DNA aptamers for ibuprofen

Single stranded DNA aptamers that can bind to ibuprofen, a widely used anti-inflammation drug, were selected from random DNA library of 10¹⁵ nucleotides by FluMag-SELEX process. Five different sequences were selected and their enantioselectivity and affinity were characterized. Three out of five aptamer candidates did not show any affinity to (S)-ibuprofen, but only to racemic form of ibuprofen, suggesting that they are (R)-ibuprofen specific aptamers. Another two aptamer candidates showed affinity to both racemic form and (S)-ibuprofen, which were considered as (S)-ibuprofen specific aptamers. The affinity of five ssDNA aptamers isolated was in a range of 1.5–5.2 μM. In addition, all of these five aptamers did not show any affinity to analogues of ibuprofen in its profen’s group (fenoprofen, flubiprofen, and naproxen) and the antibiotics of oxytetracycline, another control.     

Cryopreservation induces macrophage colony stimulating factor from human periodontal ligament cells in vitro

Cryopreservation is used to protect vital periodontal ligaments during the transplantation of teeth. We investigated which gene products implicated in root resorption are upregulated in human periodontal ligament cells by cryopreservation, and whether cryopreservation affects the expression of macrophage-colony stimulating factor (M-CSF) in human periodontal ligament cells. We used customized microarrays to compare gene expression in human periodontal ligament cells cultured from teeth immediately after extraction and from cryopreserved teeth. Based on the result of these assays, we examined M-CSF expression in periodontal ligament cells from the immediately extracted tooth and cryopreserved teeth by real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence. We also investigated whether human bone marrow cells differentiate into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts when stimulated with RANKL (Receptor Activator for Nuclear Factor κ B Ligand) together with any secreted M-CSF present in the supernatants of the periodontal ligament cells cultured from the various groups of teeth. M-CSF was twofold higher in the periodontal ligament cells from the rapid freezing teeth than in those from the immediately extracted group (p < 0.05). Cryopreservation increased M-CSF expression in the periodontal ligament cells when analyzed by real time PCR, ELISA, Western blotting, and immunofluorescence (p < 0.05). TRAP positive osteoclasts were formed in response to RANKL and the secreted M-CSF present in the supernatants of all the experimental groups except negative control. These results demonstrate that cryopreservation promotes the production of M-CSF, which plays an important role in root resorption by periodontal ligament cells.     

Sunxiuqinia dokdonensis sp. nov., isolated from deep sub-seafloor sediment

A novel facultatively anaerobic strain DH1^T was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island) in the East Sea of the Republic of Korea. The new strain was characterized using polyphasic approaches. The isolate was Gram-stain-negative, motile by gliding, non-spore-forming rods, oxidase-negative, and catalase-positive; and formed colonies of orange-red color. The NaCl range for growth was 0.5–7.0% (w/v) and no growth was observed in the absence of NaCl. The isolate grew optimally at 30°C, with 2% (w/v) NaCl and at pH 7. The cell-wall hydrolysates contained ribose as a major sugar. The DNA G+C content was 40.8 mol%. The closest related strains are Sunxiuqinia faeciviva JAM-BA0302^T and Sunxiuqinia elliptica DQHS-4^T (97.9 and 96.3% sequence similarity, respectively). The level of DNA-DNA relatedness between strain DH1^T and S. faeciviva JAM-BA0302^T was around 41% (but only 6% between DH1T and S. elliptica DQHS-4^T). The major cellular fatty acids of the isolate were contained iso-C_15:0 (25.9%), anteiso-C_15:0 (16.7%), and summed feature 9 (comprising C_16:0 3-OH and/or unknown fatty acid of dimethylacetal ECL 17.157; 13.2%). The predominant menaquinone was MK-7. On the basis of polyphasic evidence from this study, the isolate was considered to represent a novel species of the genus Sunxiuqinia, for which the name Sunxiuqinia dokdonensis sp. nov. is proposed; the type strain is DH1^T (=KCTC 32503^T =CGMCC 1.12676^T =JCM 19380^T).