Serine496 of β2 subunit of L-type Ca2+ channel participates in molecular crosstalk between activation of (Na++K+)-ATPase and the channel

Activation of (Na++K+)-ATPase (NKA) regulates cardiac L-type Ca2+ channel (LTCC) function through molecular crosstalk. The mechanism underlying NKA-LTCC crosstalk remains poorly understood. We have previously shown that activation of NKA leads to phosphorylation of LTCC α1 Ser1928. Here we investigated whether LTCC β2 subunit is modulated by NKA activation and found that LTCC β2 Ser496 is phosphorylated in response to activation of NKA. Src inhibitor PP1 and Erk1/2 inhibitor PD98059 abolish LTCC β2 Ser496 phosphorylation, suggesting that NKA-mediated β2 Ser496 phosphorylation is dependent of Src/Erk1/2 signaling pathway. Protein kinase G (PKG) inhibitor KT5823 failed to inhibit the phosphorylation of β2 Ser496, indicating that the NKA-LTCC crosstalk is independent of PKG activity. The results of nifedipine sensitive 45Ca influx experiments suggest that phosphorylation of β2 Ser496 may play a key down-regulation role in attenuating the accelerated activity of α1 subunit of the channel. Ouabain does not cause a phosphorylation on β2 Ser496, indicating a fundamental difference between activation and inhibition of NKA-mediated biological processes. This study provides the first evidence to demonstrate that LTCC β2 subunit is coupled with the movement of signals in the mechanism of activation of NKA-mediated crosstalk with LTCC.     

Identification and Characterization of GH Receptor and Serum GH-binding Protein in Chinese Sturgeon （Acipenser sinensis）

Growth hormone (GH) can stimulate bone and carti-lage cell proliferation and influence carbohydrate and lipidmetabolism. The binding of GH to its specific receptor(GHR) on the surface of target cells will induce dimeriza-tion of GHR, which allows the cytoplasmic region of GHRto interact and trigger downstream signaling and geneexpression [1,2]. GHR belongs to the cytokine receptor superfamily, andis expressed in many tissues such as the liver, muscle,adipose tissue, cartilage, and brain…     

Overexpression of Smad7 suppressed ROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1

We previously reported that AngiotensinII receptor blocker effectively inhibited TGF-β1-mediated epithelial-to-mesenchymal transition progress through regulating Smad7. However, the underlying mechanism by which Smad7 exerted in regulating MMP9 and fibrogenic response has not been fully elucidated. In the current study, we proved that NADPH p47^phox-dependent reactive oxygen species (ROS) production contributed to MMP9 activation and collagen expression, which was suppressed by transfecting pcDNA3–Smad7 in cardiac fibroblasts. The effect of Smad7 overexpression on MMP9 activity and collagen expression was further reversed by adding H₂O₂ (10 μmol/L). In contrast, knockdown of Smad7 caused the enhanced collagen synthesis in cardiac fibroblasts, which was also reversed by treating cells with a ROS inhibitor, YCG063 (2 μmol/L). Further investigation showed that Smad7 regulated NADPH-mediated ROS production through activating Heme oxygenase-1 (HO-1). Meanwhile, the intercellular level of bilirubin (product of hemin) and nitric oxide (NO) in cell supernatant were not significantly increased in cells treated with AngII or transfected with Smad7. Knockdown of HO-1 in Smad7-overexpressed cardiac fibroblasts or cells pretreated with SnPP IX, a competitive inhibitor of HO-1 activity, resulted in increased productions of ROS and NADPH p47^phox, and abolished the inhibitory effects of Smad7 on MMP9 activity and collagen expression. Our results indicated that HO-1 might be critically involved in Smad7-mediated regulation of MMP9 activity and fibrogenic genes expression via antagonizing the enhanced myocardial oxidative stress.     

Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.     

Spironolactone inhibits apoptosis in rat mesangial cells under hyperglycaemic conditions via the Wnt signalling pathway

Mesangial cells (MCs) play a crucial role in maintaining structure and function of glomerular tufts, providing structural support for capillary loops and modulating glomerular filtration by their contractility. MCs apoptosis occurs in experimental diabetic nephropathy, and this correlates with worsening albuminuria. Accumulating evidence suggests that mineralocorticoid receptor (MR) blockade effectively reduces proteinuria in diabetic nephropathy; however, it is rarely known whether spironolactone (SPI), a nonspecific MR antagonist, inhibits apoptosis in MCs under hyperglycaemic conditions. The objectives of this study are to determine the relationship between SPI and apoptosis, and investigate the cell signalling pathway by which SPI inhibits apoptosis. Rat MCs were treated with 30 mM d-glucose and 10^?8, 10^?7 or 10^?6 M aldosterone (ALD) for 24 h. In some experiments, MCs were pretreated with 10^?7 M SPI or 10 mM LiCl for 1 h. Apoptosis was evaluated by cell nucleus staining and flow cytometric analyses, and caspase-3 activity was assayed. Gene and protein expression were quantified using quantitative real-time PCR and Western blotting, respectively. SPI directly inhibited high glucose and ALD-induced MCs apoptosis in a caspase-dependent manner. Importantly, SPI inhibited MCs apoptosis via the Wnt signalling pathway. SPI promoted activation of the Wnt signalling pathway in MCs, leading to upregulation of Wnt4 and Wnt5a mRNA expression, decreased GSK-3β protein expression and increased β-catenin protein expression. As a conclusion, this study suggests that SPI may inhibit apoptosis in MCs during hyperglycaemic conditions via the Wnt signalling pathway. Blockade of the ALD system may represent a novel therapeutic strategy to prevent MCs injury under hyperglycaemic conditions.     

竹叶菜的组织培养研究

竹叶菜为百合科鹿药属一种多年生草本植物,因做菜口感好,且营养丰富、药用价值高而成为深受人们喜爱的一种野生蔬菜.有关竹叶菜及其同属的组织培养研究均未见报道.本文以竹叶菜的顶芽为外植体,通过器官发生途径,初步探索了竹叶菜的组织培养技术,结果表明:芽诱导的合适培养基为:MS +6-BA 1.5 +NAA 0.05+0.6％琼脂+3％蔗糖;芽继代培养的合适培养基为:MS+6-BA 1.2 +NAA 0.05 +0.6％琼脂+3％蔗糖;无菌小苗生根的合适培养基为:MS+ IAA 1.5+ NAA 0.2+ 0.6％琼脂+3％蔗糖.为竹叶菜今后较大规模地栽培或生产提供种苗及技术贮备,为实现竹叶菜资源的可持续性开发利用奠定了基础.     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.