Arabidopsis ; chemistry ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Intercellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Protein Conformation ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism

OBJECTIVETo investigate the role of interleukin-17 (IL-17) in alveolar fluid clearance in mice with acute lung injury (ALI) and explore the possible mechanism.

METHODSSixteen IL-17-knockout mice and 16 wild-type mice were both randomized for intratracheal instillation of PBS (control) on lipopolysaccharide (LPS) to induce ALI. Forty-eight hours after the treatments, the wet-dry ratio (W/D) of the lungs, IL-8 in the bronchoalveolar lavage fluid (BALF) and histopathological changes of the lung tissues were examined. The expressions of epithelial sodium channel α subunit (α-ENaC) was detected with Western blotting and liver kinase B1 (LKB1) was detected with immunohistochemistry.

RESULTSCompared with wild-type mice treated with LPS, IL-17 knockout mice showed significantly decreased W/D of the lungs (9.739∓3.3 vs 5.351∓0.56) and IL-8 level in the BALF (67.50∓7.33 vs 41.00∓3.16 pg/mL) following LPS challenge. Pathological examination revealed reduced alveolar edema fluid aggregations and lower lung injury score in IL-17 knockout mice with also higher expression levels of ENaC and LKB1 compared with the wild-type mice.

CONCLUSIONKnocking out IL-17 in mice not only alleviates inflammation of the lung tissue following ALI but also reduces the loss of ENaC protein and promotes alveolar fluid clearance, mechanism of which is probably associated with LKB1.

Acute Lung Injury ; metabolism ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Epithelial Sodium Channels ; metabolism ; Gene Knockout Techniques ; Interleukin-17 ; genetics ; metabolism ; Interleukin-8 ; metabolism ; Lipopolysaccharides ; Lung ; pathology ; Mice ; Protein-Serine-Threonine Kinases ; metabolism

To explore the antagonistic effect of gingerols against the inflammation induced by lectin from Pinellia ternata. In this study, ELISA method was used to determine the effect of different extracts from gingerols on the release of inflammatory factor TNF-α from macrophages induced by lectin from P. ternata. The fluorescence probe was used to determine the effect of gingerols on the changes in ROS of macrophages induced by lectin from P. ternata. The western-blot method was applied to study the effect of gingerols on the increase in expression of cell receptor interacting protein RIP3 in macrophages induced by lectin from P. ternata. The scanning electron microscope (SEM) was used to study the effect of gingerols on morphological changes in macrophages induced by lectin from P. ternata. According to the results, gingerols can significantly inhibit the release of inflammatory factor from macrophages induced by lectin from P. ternata, ROS overproduction and increase in RIP3 expression. SEM results showed that gingerols can inhibit the cytomorphosis and necrocytosis induced by lectin from P. ternata. Fresh ginger's detoxication may be related to gingerols' effects in inhibiing release of inflammatory factor, ROS overproduction and increase in RIP3 expression caused by macrophages induced by lectin from P. ternata, which are mainly inflammatory development.
Animals ; Catechols ; pharmacology ; Cells, Cultured ; Drug Antagonism ; Fatty Alcohols ; pharmacology ; Ginger ; chemistry ; Lectins ; toxicity ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Pinellia ; chemistry ; toxicity ; Reactive Oxygen Species ; metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

To observe the effects of Danshen-containing serum on SuFu and DYRK2 expression in the HSCs stimulated by leptin. SD rats (n = 60) were used to make danshen-containing serum by gastric perfusion for ten days with Danshen water decoction, normal saline and colchicine. The HSCs that were cultured in vitro would be stimulated for 24 hours by leptin (100 μg x L(-1)) except blank control group, after being intervened, the drug serum in each group would be cultured at 37 degrees C in 5% incubator. The cells would be collected after 24 hours, then the effects of danshen-containing serum on the proliferation of HSCs were detected by MTT, the expression of SuFu mRNA and DYRK2 mRNA were detected by RT-PCR, the expression of SuFu and DYRK2 proteins were tested by Western blot. Compared with blank control group, the expression of DYRK2 mRNA and DYRK2 proteins were enhanced obviously after stimulated the HSCs of rats by leptin (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, after cyclopamine group (Hh pathway inhibitor), Danshen-containing serum and colchicine were interfered, the expression of DYRK2 mRNA and DYRK2 proteins were decreased clearly (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were increased significantly (P < 0.01 or P < 0.05). Compared with model group, adding purmorphamine (Hh pathway agonist) to model group and making it activate could increase the expression of DYRK2 mRNA and DYRK2 proteins, but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, using the Danshen-containing serum to interfere the purmorphamine group could make the expression of DYRK2 mRNA and DYRK2 proteins decrease and the expression of SuFu mRNA and SuFu proteins increase significantly (P < 0.01). Danshen-containing serum would inhibition the activation and increment of HSCs by interfering the expression of SuFu and DYRK2 which were induced by leptin.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Male ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Repressor Proteins ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; Carrier Proteins ; chemistry ; metabolism ; Computational Biology ; Genes, Neoplasm ; Humans ; LIM Domain Proteins ; chemistry ; metabolism ; Mice ; Mutation ; Neoplasms ; genetics ; pathology ; Phosphoproteins ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Transferases (Other Substituted Phosphate Groups) ; chemistry ; metabolism ; Tumor Suppressor Proteins ; chemistry ; genetics ; metabolism

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

To investigate the role of the extracellular signal-regulated kinase (ERK1/2) and PI3K/AKT/ mTOR signal pathway inducing bone marrow mesenchymal stem cells (BMSCs) differentiation into neural cells, mouse bone marrow-derived mesenchymal stem cell lines D1 cells were used as research object. And they were divided into control groups and salidroside (SD) groups. Different concentrations (5, 25, 50, 100 and 200 microg x mL(-1) of SD were used and SD (100 microg x mL(-1)) was used to induce at different time (0.5, 1, 3, 6, 9, 12, 24, 48 and 72 h). The immunofluorescence staining chemical technology, real-time PCR and Western blotting were used to detect the positive rates of NSE, MAP2, beta-Tubulin III, NES, GFAP and the expression levels of beta-Tubulin III, NSE, ERK1/2, AKT. The expression of ERK1/2 and NSE was detected when the ERK1/2 and PI3K/AKT/ mTOR signal pathway was blocked by PD98059 and LY294002. It indicated that the positive rates of NSE, MAP2, beta-Tubulin III, NES and GFAP were gradually enhanced with time increased. The expression level of NSE and beta-Tubulin III protein were significantly higher than those in control groups (P < 0.01). The expression of ERK1/2, AKT mRNA and protein were higher with concentration and time increased. When the ERK1/2 and PI3K/AKT/mTOR signal pathway were blocked, the expression levels of NSE, NES and beta-Tubulin III mRNA and NSE protein were inhibited significantly. It points out that SD can stimulate the ERK1/2 and PI3K/AKT/mTOR signal pathway to promote BMSCs differentiation into neural cells.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; Glial Fibrillary Acidic Protein ; metabolism ; Glucosides ; antagonists & inhibitors ; isolation & purification ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Mesenchymal Stromal Cells ; cytology ; Mice ; Microtubule-Associated Proteins ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Morpholines ; pharmacology ; Nestin ; metabolism ; Neurons ; cytology ; metabolism ; Phenols ; antagonists & inhibitors ; isolation & purification ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphopyruvate Hydratase ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rhodiola ; chemistry ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; metabolism ; Tubulin ; metabolism

OBJECTIVETo investigate the regulative mechanism of the diterpene phenol extract of Rosmarinus Officinalis (DERO) on the imbalance of collagen metabolism of the lung tissue in pulmonary fibrosis rats.

METHODSFifty healthy Sprague-Dawley rats were randomly divided into the normal saline group (NS), the bleomycin-induced lung injury group (BLM), the low dose DERO group (at the daily dose of 50 mg/kg), the moderate dose DERO group (at the daily dose of 100 mg/kg), and the high dose DERO group (at the daily dose of 200 mg/kg), 10 in each group (abbreviated as DERO 1, 2, 3, respectively). The pulmonary fibrosis rat model was prepared by disposable intratracheal instillation of bleomycin. DERO was administered by gastrogavage as intervention during the repairing process of lung injury. On the morning of the 29th day, the rats' lung tissue was extracted. The karyocyte number, collagen protein, type I collagen (collagen I) and transforming growth factor-beta type II receptor (TGFbetaR II), Smad4 mRNA expressions were semi-quantitatively determined using tissue microarray, HE staining, collagen fiber dyeing, immunohistochemical assay, and in situ hybridization. Using real-time fluorescent quantification RT-PCR, the mRNA expression of transforming growth factor-beta1 (TGF-beta1) were detected.

RESULTSCompared with the NS group, the collagen deposition of the lung tissue was obvious and the inflammatory infiltration was more severe in the BLM group (P < 0.05, P < 0.01). There was no statistical difference in the aforesaid 4 indices between the DERO1 group and the BLM group (P > 0.05). The collagen deposition and the inflammatory infiltration were obviously alleviated in the DERO2 and DERO3 groups (P < 0.05, P < 0.01). Compared with the NS group, the mRNA expressions of collagen-I, TGF-beta1 R II, Smad4, and TGF-beta1 were obviously up-regulated in the BLM group (P < 0.05, P < 0.01). Compared with the BLM group, the aforesaid four indices were not statistically changed in the DERO1 group (P > 0.05). But the mRNA expressions of collagen-I, TGF-beta1 R II, Smad4, and TGF-beta1 were obviously downregulated in the DERO2 and DERO3 groups (P < 0.05, P < 0.01). But the down-regulation of Smad4 expression was not obvious in the DERO2 and the DERO3 groups (P > 0.05). Compared with the DERO1 group, the mRNA expressions of collagen-I, TGF-beta1, R II, TGFbeta1 were all obviously lower in the DERO2 and the DERO3 groups (P < 0.05). But there was no statistical difference in the aforesaid 4 indices between the DERO2 group and the DERO3 group (P > 0.05).

CONCLUSIONSDERO could regulate imbalanced collagen metabolism of pulmonary fibrosis. It could inhibit excessive deposition of collagen fibers, especially excessive deposition of collagen- I. Its mechanisms might be realized by inhibiting up-regulation of TGF-beta1 and TGFbetaR II mRNA expressions, thus interfering the activation of TGF-beta-Smad signaling pathway on target genes, especially on type I procollagen target gene.

Animals ; Collagen Type I ; metabolism ; Diterpenes ; pharmacology ; Female ; Lung ; drug effects ; metabolism ; Male ; Plant Extracts ; pharmacology ; Protein-Serine-Threonine Kinases ; metabolism ; Pulmonary Fibrosis ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; metabolism ; Rosmarinus ; chemistry ; Signal Transduction ; Transforming Growth Factor beta1 ; metabolism

Under natural environments, plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions. The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane. A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and long-term response. Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp. RCC299 (MsStt7d) in the apo form and in complex with various nucleotide substrates. MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site. A novel hairpin motif, found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability, interacts with the activation loop and fixes it in an active conformation. We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues. Moreover, preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly. The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer. The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.
Amino Acid Sequence ; Amino Acid Substitution ; Arabidopsis ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Binding Sites ; Catalytic Domain ; Chlorophyta ; enzymology ; Crystallography, X-Ray ; Cyclin-Dependent Kinase 2 ; chemistry ; metabolism ; Molecular Sequence Data ; Phosphorylation ; Protein Structure, Secondary ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA-PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.
Actins/*metabolism ; Apoptosis ; Catalytic Domain ; Cell Cycle Proteins/genetics/*metabolism ; Cell Line ; Cell Line, Tumor ; DNA-Activated Protein Kinase/chemistry/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Furans/pharmacology ; *G1 Phase ; Gene Knockdown Techniques ; Humans ; Phosphorylation/drug effects ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; Pyrans/pharmacology ; Tumor Suppressor Protein p53/*metabolism ; Tumor Suppressor Proteins/genetics/*metabolism