OBJECTIVES: To investigate the molecular mechanism by which salvianolic acid B (Sal-B) modulates mitochondrial functional homeostasis and alleviates myocardial ischemia-reperfusion (I/R) injury in mice. METHODS: Mouse cardiomyocyte HL-1 cells were pretreated with 5 μmol/L Sal-B with or without sh-Sirt1 transfection before exposure to hypoxia-reoxygenation (HR), and the changes in ATP production, mitochondrial superoxide activity, substrate oxidation level were evaluated. In the animal experiment, 36 C57BL/6J mice were randomized into 3 groups (n=12) for sham operation or ligation of the left anterior coronary artery to induce myocardial I/R injury with or without intravenous injection of Sal-B+I/R (50 mg/kg). In the rescue experiment, 60 adult C57BL/6J mice were randomized into 5 groups (n=12): sham-operated group, myocardial I/R group, Sal-B+I/R group, I/R+Sal-B+Sirt1fl/fl group, and I/R+Sal-B+cKO-Sirt1 group. Myocardial injury was evaluated with HE staining, and cardiac function was assessed by measurement of the ejection fraction and fractional shortening using echocardiography. RESULTS: In HL-1 cells with HR injury, Sal-B pretreatment significantly increased cellular ATP production, reduced mitochondrial superoxide anion levels, and enhanced oxygen consumption level. In the mouse models of myocardial I/R injury, Sal-B pretreatment markedly ameliorated I/R-induced structural disarray of the cardiac myocytes and improved cardiac ejection. Cycloheximide chase with Western blotting and ubiquitination assays after Sirt1-IP showed that Sal-B significantly inhibited Sirt1 degradation in HL-1 cells. Sirt1 knock-down reversed Sal-B-induced increases in ATP production, reduction in superoxide, and elevation of OCR in HL-1 cells. Cardiomyocyte-specific Sirt1 knockout obviously reversed Sal-B-mediated improvement in cardiac ejection function and myocardial structure damage in mice with myocardial I/R injury. CONCLUSIONS Sal-B promotes mitochondrial functional homeostasis in cardiomyocytes with HR injury and improves cardiac function in mice after myocardial I/R by inhibiting Sirt1 protein degradation.
Animals ; Sirtuin 1/metabolism* ; Myocardial Reperfusion Injury/physiopathology* ; Mice, Inbred C57BL ; Mice ; Myocytes, Cardiac/drug effects* ; Benzofurans/pharmacology* ; Homeostasis/drug effects* ; Male ; Mitochondria/drug effects* ; Depsides

Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.
Apoptosis ; drug effects ; Azepines ; pharmacology ; Cell Differentiation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; genetics ; Nuclear Proteins ; genetics ; Promyelocytic Leukemia Protein ; genetics ; RNA, Messenger ; genetics ; Retinoic Acid Receptor alpha ; genetics ; Transcription Factors ; genetics ; Triazoles ; pharmacology ; Tumor Cells, Cultured

S100A9,a calcium-binding protein,participates in the inflammatory process and development of various tumors,thus attracting much attention in the field of cancer biology.This study aimed to investigate the regulatory mechanism of S100A9 and its function involvement in APL.We used real-time quantitative PCR to determine whether PML/RARα affects the expression of S100A9 in NB4 and PR9 cells upon ATRA treatment.ChiP-based PCR and dual-luciferase reporter assay system were used to detect how PML/RARα and PU.1 regulate S100A9 promoter activity.CCK-8 assay and flow cytometry were employed to observe the viability and apoptosis of NB4 cells when S100A9 was overexpressed.Results showed that S100A9 was an ATRA-responsive gene,and PML/RARα was necessary for the ATRA-induced expression of S100A9 in APL cells.In addition,PU.1 could bind to the promoter of S100A9,especially when treated with ATRA in NB4 cells,and promote its activity.More importantly,overexpression of S100A9 induced the apoptosis of NB4 cells and inhibited cell growth.Collectively,our data indicated that PML/RARα and PU.1 were necessary for the ATRA-induced expression of S100A9 in APL cells.Furthermore,S100A9 promoted apoptosis in APL cells and affected cell growth.

OBJECTIVETo investigate the phenotype-genotype association of isodicentromere Y chromosome by analysis of two female patients carrying the chromosome with sexual development disorders.

METHODSThe karyotypes of the two patients were determined by application of conventional G banding of peripheral blood samples and fluorescence in situ hybridization (FISH). PCR was applied to detect the presence of SRY gene.

RESULTSConventional karyotype analysis showed case 1 to be a mosaic: mos.45,X[38]/46,X,+mar[151]/47,XY,+mar[5]/47,X,+mar × 2[2]/46,XY[4], FISH showed that 12 different cell lines were presented in the karyotype of case 1 and partial cell lines with SRY gene, the marker is an isodicentromere Y chromosome [idic(Y)(p)]. No mutation was found in the SRY gene. The karyotype of case 2 was mos.45,X[25]/46,X,+mar[35]. FISH showed the marker to be an idic(Y)(p) without the SRY gene.

CONCLUSIONThe karyotype of patients carrying idic(Y)(p) seems unstable, and female patients have the characteristics of short stature and secondary sexual hypoplasia. Karyotype analysis combined with FISH analysis can accurately determine the breakpoint of idic(Y) and identify the types of complex mosaic, which may facilitate genetic counseling and prognosis.

Adolescent ; Child ; Chromosomes, Human, Y ; Disorders of Sex Development ; genetics ; Female ; Humans ; Karyotype ; Sex Chromosome Aberrations ; Sex-Determining Region Y Protein ; genetics

Objective To establish several human umbilical vein endothelial cell ( HUVEC ) strains with over-ex-pression or low expression of receptor for activated C kinase 1 ( RACK1 ) , which will provide an effective tool for future studying the function of RACK1 in arrhythmia.Methods The full-length cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-EGFP.At the same time, designed and synthesised complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence , then subcloned into the plas-mid pGenesil-1 .The HUVEC cells were transfected with these plasmids and screened by using G 418 .And the expression of RACK1 mRNA and protein in the cells were assayed by qRT-PCR and Western blot , respectively . Results RACK1 eukaryotic expression vector and siRNA expression vectors of RACK 1 were constructed success-fully.After a 48 h transfection of HUVEC cells with the recombinant vectors and G 418 selection, the positive cell clones were obtained .qRT-PCR and Western blot showed that over-expression vector and interference vectors could effectively enhanced and knocked-down RACK1 expression in HUVEC strains .Conclusions HUVEC cell strains with over-expression and low expression of RACK 1 have been successfully established .

Objective_To investigate the potential effect of bone marrow mesenchymal stem cells ( MSCs) on repai-ring the colon epithelial cell,and on the treatment of rats with ulcerative colitis ( UC) .Methods_Monocytes were purified from bone marrow, amplified and identified as MSCs in vitro.Thirty female Wistar rats were randomly di-vided into 3 groups, the normal control, model and MSCs groups (10 rats/group).The rats in model and MSCs groups were induced colitis with trinitro-benzene-sulfonic acid;The rats in normal control group and model groups were injected with 1mL saline via tail vein, while those in MSCs group with 1 mL MSCs suspension.After two weeks, colon tissue samples were analyzed for histopathology,and the colon tissues were made into serial section for determining the distribution of Y chromosome and CK20 double positive cells,analyzing the mRNA levels of CK20,NF-κB, IL-4 by RT-PCR,and assaying colonic NF-κB protein expression with Westen blot,detecting colon tissues IL-4 content with ELISA.Results_Y chromosome and CK20 double positive cells were found in MSCs transplanted colon tissues.The expression of CK20 increased in the colon tissues of UC rats(P<0.01) and in MSCs group in-creased as compared with model group( P<0.01) .The expression of NF-κB increased in the colon tissues of UC rats (P<0.01), but decreased MSCs group as compared with model group (P<0.01).The expression of IL-4 was decreased in the colon tissues of UC rats ( P<0.01) , while in MSCs group it was increased as compared with model group ( P<0.01) .Conclusions_MSCs may have therapeutic efficacy on colitis in rats through differentiating into colon epithelial cells.

OBJECTIVETo investigate the clinical and molecular genetics characteristics of a patient with mild androgen insensitivity syndrome (MAIS).

METHODSClinical data of the patient was collected, and DNA was isolated from peripheral blood sample. Eight exons of AR gene were amplified by PCR with specific primers and directly sequenced by Sanger method. The results were compared with standard sequences from GenBank. Online Polyphen-2 software was applied to predict the effect of mutation on the protein function and compare the conservation of the sequence at the mutation site in various species. The exon of the AR gene containing the mutated site was analyzed in 90 unrelated normal males using PCR and restrictive digestion with Sfa NI.

RESULTSSequence analysis has detected a novel missense mutation in codon 176 of exon 1 (Ser176Arg) of the AR gene. Analysis with polyphen-2 software has indicated the codon to be highly conserved across various species, and that the S176A mutation has caused damage to the protein structure and function (prediction score=0.999). The same mutation was not detected in 90 healthy males.

CONCLUSIONThe S176A mutation of the AR gene may contribute to the mild androgen insensitivity syndrome.

Adult ; Amino Acid Sequence ; Androgen-Insensitivity Syndrome ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Receptors, Androgen ; genetics

OBJECTIVETo determine the molecular etiology for a muscular dystrophy pedigree with target region sequencing platform using hereditary myopathy capture array.

METHODSSpecific gene testing was performed based on the clinical diagnosis. Since no pathogenic mutation was found, target region sequencing with hereditary myopathy capture array combined with Sanger sequencing and bioinformatics analysis were employed in turn. PolyPhen and NCBI were used to evaluate the pathogenicity of identified mutation and conservation of the gene.

RESULTSTarget region sequencing indicated the proband has carried a heterozygous c.3353 A>C mutation of COL6A3 gene, which was confirmed by Sanger-sequencing in 4 affected individuals from the family. The same mutation was not detected in healthy members of the pedigree. Bioinformatics analysis suggested that the mutation has caused a highly pathogenic amino acid substitution from Histidine to Proline. The affected patients featured normal intelligence with mild myogenic damage by muscle biopsy, slightly increased serum creatine kinase and slow disease progression, which was consistent with Bethlem myopathy.

CONCLUSIONTarget region sequencing is an effective and efficient method for genetic testing. The heterozygous c.3353A>C mutation in exon 8 of the COL6A3 gene probably underlies the Bethlem myopathy with autosomal dominant inheritance.

Adult ; Amino Acid Sequence ; Amino Acid Substitution ; Base Sequence ; Collagen Type VI ; genetics ; Contracture ; genetics ; Exons ; Female ; Heterozygote ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Muscular Dystrophies ; congenital ; genetics ; Mutation, Missense ; Pedigree ; Young Adult