OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

As an important industrial chemical, isoprene is mainly used as a precursor for synthetic rubbers. In addition, it also has wide applications in the field of pharmaceutical and chemical intermediates, food, adhesives and aviation fuel. Compared with conventional petrochemical routes, production of isoprene in microbial systems has been the research focus considering environment friendly and sustainable development features. This article summarizes the metabolic pathways and key enzymes of isoprene biosynthesis, reviews current methods and strategies in improving isoprene production of Escherichia coli, and also gives some basic ideas and expectation.
Butadienes ; Escherichia coli ; Hemiterpenes ; biosynthesis ; Industrial Microbiology ; Metabolic Engineering ; Metabolic Networks and Pathways ; Pentanes

Isoprene is an important precursor of synthetic rubber material. In our previous study, metabolic engineered Escherichia coli strain (BW-01) was constructed and used to produce isoprene. Based on the theory of protein budget, using synthetic biology strategies including the increased copy number of genes and rare codons, we regulated the expression of key enzyme to improve isoprene production in Escherichia coli strain. Under shake-flask conditions, isoprene productivity of the engineered strain (BW-07) increased by 73% compared with BW-01, reached 761.1 mg/L. It provides a reference for further studies.
Butadienes ; Escherichia coli ; genetics ; metabolism ; Gene Dosage ; Hemiterpenes ; biosynthesis ; Industrial Microbiology ; Metabolic Engineering ; Mevalonic Acid ; Pentanes ; Synthetic Biology

OBJECTIVETo investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in the regulation of aquaporin 4 (AQP4) expression in cultured astrocytes after scratch-injury.

METHODSThe scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase (LDH) leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2 (p-ERK1/2) expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 µmol/L U0126 (ERK1/2 inhibitor) was incubated in the medium at 30 min before the scratch-injury in some groups.

RESULTSIncreases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury.

CONCLUSIONThese results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Animals ; Aquaporin 4 ; metabolism ; Astrocytes ; enzymology ; metabolism ; Butadienes ; administration & dosage ; Cells, Cultured ; Down-Regulation ; Enzyme Activation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; MAP Kinase Signaling System ; Nitriles ; administration & dosage ; Rats ; Rats, Wistar ; Skin ; injuries

Air ; analysis ; Alkenes ; analysis ; Butadienes ; analysis ; Chromatography, Gas ; methods ; Environmental Monitoring ; methods ; Workplace

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.
Ascorbic Acid ; pharmacology ; Butadienes ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Co-Repressor Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; Phosphorylation ; drug effects ; RNA Interference ; RNA, Small Interfering ; metabolism ; Stem Cells ; cytology ; Transcription Factors ; antagonists & inhibitors ; genetics ; metabolism ; Up-Regulation ; drug effects

Connective tissue growth factor (CTGF) is a potent pro-fibrotic factor, which is implicated in fibrosis through extracellular matrix (ECM) induction in diabetic cardiovascular complications. It is an important downstream mediator in the fibrotic action of transforming growth factor beta (TGFbeta) and is potentially induced by hyperglycemia in human vascular smooth muscle cells (VSMCs). Therefore, the goal of this study is to identify the signaling pathways of CTGF effects on ECM accumulation and cell proliferation in VSMCs under hyperglycemia. We found that high glucose stimulated the levels of CTGF mRNA and protein and followed by VSMC proliferation and ECM components accumulation such as collagen type 1, collagen type 3 and fibronectin. By depleting endogenous CTGF we showed that CTGF is indispensable for the cell proliferation and ECM components accumulation in high glucose-stimulated VSMCs. In addition, pretreatment with the MEK1/2 specific inhibitors, PD98059 or U0126 potently inhibited the CTGF production and ECM components accumulation in high glucose-stimulated VSMCs. Furthermore, knockdown with ERK1/2 MAPK siRNA resulted in significantly down regulated of CTGF production, ECM components accumulation and cell proliferation in high glucose-stimulated VSMCs. Finally, ERK1/2 signaling regulated Egr-1 protein expression and treatment with recombinant CTGF reversed the Egr-1 expression in high glucose-induced VSMCs. It is conceivable that ERK1/2 MAPK signaling pathway plays an important role in regulating CTGF expression and suggests that blockade of CTGF through ERK1/2 MAPK signaling may be beneficial for therapeutic target of diabetic cardiovascular complication such as atherosclerosis.
Animals ; Aorta ; Atherosclerosis ; Butadienes ; Cell Proliferation ; Collagen ; Connective Tissue ; Connective Tissue Growth Factor ; Diabetes Complications ; Extracellular Matrix ; Fibronectins ; Fibrosis ; Flavonoids ; Glucose ; Humans ; Hyperglycemia ; Muscle, Smooth, Vascular ; Nitriles ; Phosphotransferases ; Rats ; RNA, Messenger ; RNA, Small Interfering ; Transforming Growth Factor beta

To develop a simple, validated method for identifying and quantifying 1,3-butadiene (BD) in human blood by gas chromatography-mass spectrometry (GC-MS) and head-space gas chromatography (HS-GC). BD was identified by GC-MS and HS-GC, and quantified by HS-GC. The method showed that BD had a good linearity from 50 to 500 microg/mL (r > 0.99). The limits of detection and quantification were 10 microg/mL and 50 microg/mL, respectively. Both the intra-day precision and inter-day precision were < 6.08%, and the accuracy was 96.98%-103.81%. The method was applied to an actual case, and the concentration of BD in the case was 242 microg/mL in human blood. This simple method is found to be useful for the routine forensic analysis of acute exposure to BD.
Adult ; Butadienes/poisoning* ; Forensic Toxicology/methods* ; Gas Chromatography-Mass Spectrometry/methods* ; Gas Poisoning ; Humans ; Male ; Reproducibility of Results ; Sensitivity and Specificity ; Solvents/chemistry* ; Temperature

Linalool is an important monoterpene, and widely used in food, pharmaceutical and cosmetic industry. The low concentration in plants and the difficulties in extraction restrict its large scale production. Saccharomyces cerevisiae can provide the monoterpene precursor, geranyl diphosphate (GPP) through its endogenous isoprenoid pathway. Therefore, it could be used as the host for monoterpene production. However, the weak metabolic flux through the isoprenoid pathway leads to the insufficient supply of GPP, and results in low monoterpene productivity. In order to increase the metabolic flux, we constructed the integrated expression plasmid pRS305-tHMG1 and free expression plasmid pYLIS-IDI1 to enhance the expression levels of isopentenyl diphosphate isomerase (IDI1) and a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMG1). The two plasmids were separately transformed into S. cerevisiae CEN.PK2-1C, resulting in strains LS01 and LS02. The plasmid pYLIS-IDI1 was further transformed into strain LS01, resulting in strain LS03. GC-MS analysis showed that the linalool concentration was increased by 1.3 times and reached (127.71 +/- 7.68) microg/L. In conclusion, enhancement of the supply of GPP precursors through the regulation of isoprenoid pathway could increase the linalool production in S. cerevisiae.
Biosynthetic Pathways ; genetics ; Butadienes ; metabolism ; Hemiterpenes ; metabolism ; Monoterpenes ; metabolism ; Pentanes ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism

BACKGROUNDInvasive aspergillosis (IA), which is mainly caused by Aspergillus fumigatus (A. fumigatus), is a major cause of morbidity and mortality in immunocompromised patients. Despite considerable progress in currently available antifungals the mortality still remains high in critically ill patients. U0126 which is a highly selective inhibitor of MEK1 and MEK2 in the RAF/MEK/ERK pathway in mammalian cells has been demonstrated to have an anti-proliferative role in cancer cells. The purpose of this study was to explore the role of U0126 on growth inhibition and activation of mitogen-activated protein kinases (MAPKs) in A. fumigatus.

METHODSGermination percentage and hyphae growth in A. fumigatus treated with U0126 were observed and compared with untreated controls. Western blotting analysis was used to detect changes in activation of SakA, MpkA and MpkB.

RESULTSU0126 inhibited germination and hyphae growth in A. fumigatus and enhanced the phosphorylation of SakA and MpkA under oxidative stress. U0126 at 10 µmol/L did not block the activation of MpkB during nitrogen starvation stress.

CONCLUSIONU0126 shows promise as an antifungal candidate and the MAPK pathway may be a possible antifungal drug target for A. fumigatus.

Aspergillus fumigatus ; drug effects ; enzymology ; growth & development ; Butadienes ; pharmacology ; Enzyme Activation ; drug effects ; Enzyme Inhibitors ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinases ; metabolism ; Nitriles ; pharmacology