Objective of this study was to investigate the transcriptional regulation of BHLHB2 gene by the PML-RARα fusion protein in APL cells and reveal the pathogenesis of APL. RT-PCR was performed to detect the expression change of BHLHB2 before and after the induction of PML-RARα in PR9 cells, and its expression level after the treatment of ATRA in PR9 and APL patient derived NB4 cells. Chromatin immunoprecipitation (ChIP)-based PCR was used to analyze whether the BHLHB2 promoter could be bound by PML-RARα in vivo. A large-scale gene expression profile dataset was used to observe the expression pattern of BHLHB2 in AML. The results showed that the expression level of BHLHB2 was significantly reduced with the induction of PML-RARα and ATRA could reverse this inhibition in both PR9 and NB4 cells and increase the expression of BHLHB2. However, the expression of BHLHB2 could not be induced by ATRA in U937 cells which do not express PML-RARα. Mechanism study revealed that PML-RARα could bound to the promoter of BHLHB2 in vivo to regulate the the expression of BHLHB2. It was found that the expression of BHLHB2 was relatively lower in APL as compared with other subtypes of AML and normal bone marrow cells. It is concluded that BHLHB2 is the target of PML-RARα, and the expression of BHLHB2 is inhibited by PML-RARα through binding to its promoter in APL.
Basic Helix-Loop-Helix Transcription Factors ; genetics ; Gene Expression Regulation, Leukemic ; Homeodomain Proteins ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; pathology ; Oncogene Proteins, Fusion ; genetics ; Promoter Regions, Genetic ; Transcription Factors ; genetics ; Tumor Cells, Cultured ; U937 Cells

To investigate and compare the expression of intercellular adhesion molecule-1 (ICAM-1) in different organs of the mice with endotoxic shock induced by lipopolysaccharide (LPS), protein and mRNA of ICAM-1 were measured by Western blotting and RT-PCR respectively in different organs of BALB/c mice administered intraperitoneally with 5 mg/kg LPS. The results showed that the constitutive expression of ICAM-1 protein and mRNA was the greatest in the lungs, followed by the spleen, kidney and intestine. After LPS stimulation, the upregulation of ICAM-1 was still greatest in the lungs, followed by the liver, spleen, heart, kidney and intestine. Compared with the normal mice, the expression of ICAM-1 protein in endotoxic shocked mice increased by 4.5-fold in the lungs, 3.0-fold in the kidney, 1.5-fold in the spleen; the expression in the liver and heart was negative under normal condition and changed into positive during endotoxic shock; but ICAM-1 expression in the intestine did not change significantly. The expression of ICAM-1 mRNA also increased consistently. These data highlight that LPS can up-regulate ICAM-1 protein and mRNA expression in different tissues of the mice with endotoxic shock. The difference in ICAM-1 expression among the organs may lead to different sensitivity of organ damage in endotoxic shock. This suggests that inhibition of ICAM-1 expression may be a useful principle for prevention and treatment of endotoxic shock.
Animals ; Intercellular Adhesion Molecule-1 ; biosynthesis ; Kidney ; metabolism ; Lipopolysaccharides ; Lung ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; Shock, Septic ; chemically induced ; metabolism

BACKGROUNDAdvanced oxidation protein products (AOPPs) are new uremic toxins reported by Witko-Sarsat in 1996, which are associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of AOPPs on MCP-1 expression in cultured vascular smooth muscle cells (VSMCs).

METHODSVSMCs were cultured and then co-incubated with AOPP (200 micromol/L, 400 micromol/L) for different times with or without pretreatment with specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. RT-PCR and Western blott were used to detect MCP-1 mRNA and protein expression at different time points after AOPP stimulation in rat smooth muscle cells. Western blot was used to detect the expression of phosphorylated p38 MAPK.

RESULTSTreatment of VSMC with AOPPs resulted in a significant increase of the expression of MCP-1 mRNA and protein in time- and dose-dependent manner, and could activated p38 MAPK. Pretreatment of VSMCs with SB203580 resulted in a dose-dependent inhibition of AOPPs-induced MCP-1 mRNA and protein expression.

CONCLUSIONSAOPPs can stimulate MCP-1 expression via p38 MAPK in VSMCs. This suggests that AOPPs might contribute to the formation of atherosclerosis through this proinflammatory effect.

Animals ; Atherosclerosis ; etiology ; Cardiovascular Diseases ; etiology ; Cells, Cultured ; Chemokine CCL2 ; genetics ; Enzyme Activation ; Imidazoles ; pharmacology ; Kidney Failure, Chronic ; complications ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Oxidation-Reduction ; Proteins ; metabolism ; Pyridines ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Uremia ; metabolism ; p38 Mitogen-Activated Protein Kinases ; physiology

FGF21 (fibroblast growth factor 21) is a recently described member of the FGF family. It has been previously demonstrated that FGF21 is a potent regulator of glucose homeostasis. To improve stability of FGF21 for better efficacy, a new form of recombinant FGF21 was generated by fusion of a full length FGF21 gene and the Fc fragment of human IgG4 with flexible linker sequence. To examine the glucose regulation activity of FGF21-L-Fc, 3T3-L1 pre-adipocytes were differentiated into adipocytes, and glucose uptake activity of FGF21-L-Fc was examined by glucose oxidase and peroxidase (GOD-POD) assay. The results showed that in comparison with wild type FGF21, FGF21-L-Fc was more potent in stimulation of glucose uptake by 3T3-L1. In vivo studies on the modified protein demonstrated that FGF-L-Fc had a better efficacy in lowering blood glucose of the STZ-induced diabetic animals and controlled glucose level for a longer time. The results provided a sound basis for further studies.
3T3-L1 Cells ; Adipocytes ; metabolism ; Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Escherichia coli ; metabolism ; Fibroblast Growth Factors ; genetics ; pharmacology ; Glucose ; metabolism ; Immunoglobulin G ; genetics ; pharmacology ; Male ; Mice ; Recombinant Proteins ; genetics ; pharmacology

OBJECTIVETo detect 22q11 microdeletion in the children and fetuses affected by congenital heart defects.

METHODMLPA P250 kit was used to detect 22q11 microdeletion in 100 cases of sporadic congenital heart defects including 40 fetuses and 60 patients diagnosed by ultrasound.

RESULTTwo cases from the fetuses and 1 case from the patients were found to have 22q11 microdeletion.

CONCLUSIONThree cases had 22q11 microdeletion in the congenital heart defects.

Child ; Child, Preschool ; Chromosome Deletion ; Chromosomes, Human, Pair 22 ; Female ; Heart Defects, Congenital ; diagnosis ; genetics ; Humans ; Infant ; Male ; Nucleic Acid Amplification Techniques ; methods

OBJECTIVETo construct a recombinant adenovirus vector expressing hCD40L gene and explore it in the use of anti-tumor gene therapy.

METHODS1,900 bp gene fragment was obtained form plasmid pORF-hCD40L by Xho I/Swa I cutting and then cloned directionally into the pShuttle plasmid, finally, the resultant plasmid was digested by restriction endonnuclease PmeI and subsequently cotransformtion into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant and then the recombinant was packaged in the 293 cells. Some methods such as PCR and endonulease digestion were employed to identify the recombinant adenovirus.

RESULTSThe evidences of endonulease digestion and PCR analysis confirmed that recombinant hCD40L gene was correctly inserted into adenovirus vector.

CONCLUSIONThe adenoviral vector which expressed hCD40L gene was constructed. It provides an experimental basis for studies on it expression in the mammalian cells and in tumor gene therapy.

Adenoviridae ; Adenoviruses, Human ; Animals ; CD40 Ligand ; Genetic Therapy ; Genetic Vectors ; Humans ; Plasmids ; Polymerase Chain Reaction

OBJECTIVETo silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique.

METHODSThe vector containing the human U6 promoter was used for targeted gene silencing when a dsDNA oligonucleotide encoding an appropriate shRNA was ligated into the vector, and 67nt oligonucleotide fragment was inserted into the downstream of the U6 promoter. Plasmids containing different Raf-1 target sequences [ (1) pshuttle-Raf-1-a( 225), (2) pshattle-Raf-1-b ( 358) and (3) pshuttle-Raf-1-c(474)], were transfected into HNE1 cells. Expression of Raf-1 mRNA was assayed by RT-PCR. Apoptosis were determined by cytometry.

RESULTSVector-based RNAi had advantages over antisense RNA because it could be delivered to the target cell more efficiently, and effect could last longer. Raf-1 expression could be inhibited by plasmid-expressed shRNA. Three different targeting sequences were selected from Raf-1 gene, and the inhibitory effect of pSIREN shuttle-Raf-1-b (358) was biggest.

CONCLUSIONRaf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.

Apoptosis ; genetics ; Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-raf ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo evaluate the significance of somatic mutations of VHL gene and hypoxia-inducible factor-1alpha (HIF-1alpha) expression in primary renal clear cell carcinoma (RCC).

METHODSMutation of VHL gene and HIF-1alpha expression were detected by means of PCR, denaturing high-performance liquid chromatography (DHPLC), direct sequencing and immunohistochemistry in 32 samples from primary renal clear cell carcinoma patients.

RESULTSIn 32 RCC samples, 17 samples (53.1%) had and 32 samples of adjacent nonmalignant renal tissue had not mutations of VHL gene expression. Twelve RCC samples (70.6%) which had mutations of VHL gene expressed HIF-1alpha, and it had significant difference to 4 RCC (26.7%) samples which didn't have mutations of VHL gene (P < 0.05).

CONCLUSIONMutations of VHL gene may play a significant role in the tumorigenesis of RCC, and HIF-1alpha expression correlates with it.

Adenocarcinoma, Clear Cell ; genetics ; pathology ; Adult ; Aged ; Carcinoma, Renal Cell ; genetics ; pathology ; Chromatography, Liquid ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Immunohistochemistry ; Kidney ; chemistry ; metabolism ; pathology ; Kidney Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Mutation ; genetics ; Polymerase Chain Reaction ; Transcription Factors ; analysis ; genetics ; Tumor Suppressor Proteins ; analysis ; genetics ; Ubiquitin-Protein Ligases ; analysis ; genetics ; Von Hippel-Lindau Tumor Suppressor Protein

Objective To investigate the curative effect and mechanism of α lipoic acid combined with epalrestat and methylcobalamin in patients with type 2 diabetes mellitus (T2DM) complicated with pe-ripheral neuropathy ( DPN) . Methods A total of 160 cases of patients with DPN were randomly divided into the control group ( treated with methylcobalamin and epalrestat) and the observation group ( treated with methylcobalamin, epalrestat and α lipoic acid) , and all patients were treated for 4 weeks. The therapeutic effect, nerve conduction velocity, oxidative stress index and related proteins expression in serum were ob-served in two groups. Results The effective rate of treatment in the observation group was higher than that in the control group (87. 50％ vs 75. 0％) (χ2 = 4. 103,P<0. 05). After treatment, the sensory nerve conduction velocity ( SNCV) and motor nerve conduction velocity ( MNCV) of median nerve and common peroneal nerve were significantly better in the observation group than the control group ( P<0. 05 ) . After treatment, the level of superoxide dismutase ( SOD) was significantly higher in the observation group than the control group, while the level of malondialdehyde ( MDA ) was significantly lower the control group (P<0. 05). After treatment, the level of Bcl-2 protein was significantly higher in observation group than the control group, while the levels of Bax and Caspase-3 proteins were significantly lower than the control group (P<0. 05). Conclusions The application ofαlipoic acid combined to epalrestat and methylcobal-amin in the treatment of DPN can significantly improve the sensory and motor nerve conduction.

OBJECTIVETo establish an assay for screening chromosome 22q11 microdeletion efficiently, and apply it for detecting del22q11 in patients with non-syndromic congenital heart defects (CHD).

METHODSSeventy nine patients with non-syndromic CHD and 84 normal controls were genotyped for 8 short tandem repeat (STR) markers located in 22q11 region, by using quantitative fluorescence polymerase chain reaction (QF-PCR).

RESULTSThe average heterozygosity of the STR markers in patients and controls was 0.76 and 0.79, respectively. One patient with Tetralogy of Fallot (TOF) from the 79 CHD cases (1.3%) was found to have a deletion within chromosome 22q11.2, which was confirmed by multiplex ligation-dependent probe amplification (MLPA).

CONCLUSIONThe QF-PCR assay developed in this study was a reliable and an efficient alterative approach to screen for 22q11 microdeletion in clinical diagnosis and genetic counseling.

Case-Control Studies ; Chromosome Deletion ; Chromosomes, Human, Pair 22 ; genetics ; Fluorescence ; Heart Defects, Congenital ; diagnosis ; genetics ; Humans ; Microsatellite Repeats ; Polymerase Chain Reaction ; instrumentation ; methods