OBJECTIVETo investigate the impact of ethanol extracts from Sedum sarmentosum (ESB) on STAT-3 signaling and its probable molecular mechanism in inducing apoptosis.

METHODMTT assay was used to detect the impact of ESB on HepG2 cell proliferation. FITC-Annexin V-FITC /PI double-labeling were used to investigate the impact on hepatoma carcinoma cell apoptosis. Western blot analysis was used to test the expression levels of cell apoptosis-related proteins Caspase-3, Caspase-9, PARP, P-STAT-3 (Tyr705) , STAT-3, Bcl-2, Mcl-1.

RESULTESB could notably inhibit proliferation of HepG2 cells, and induce HepG2 cell apoptosis, with the dose-dependent inhibitory effect. In addition, ESB could inhibit STAT-3 signaling, down-regulate Mcl-1 and Bcl-2 expressions, and induce degradation/activation of apoptosis-related proteins Caspase-3 and Caspase-9 and PARP degradation in a dose-dependent manner.

CONCLUSIONESB inhibits HepG2 cell proliferation and induces apoptosis by inhibiting STAT-3 signaling and Mcl-1 and Bcl-2 expressions.

Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Ethanol ; chemistry ; Flow Cytometry ; Hep G2 Cells ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Plant Extracts ; chemistry ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Sedum ; chemistry ; Signal Transduction ; drug effects ; Time Factors

italic>Alangium chinense is a commonly used medicinal plant of Alangiaceae Alangium, one of the characteristic Miao medicines in Guizhou. The genus is strongly differentiated and has complex morphological variation, with significant differences in active ingredients and potency among herbs. In this paper, we sequenced the chloroplast genomes of Alangium chinense subsp. Pauciflorum, Alangium chinense subsp. Strigosum and Alangium kurzii Craib using Illumina high-throughput sequencing technology. The genomes of Alangium chinense subsp. Pauciflorum, Alangium chinense subsp. Strigosum and Alangium kurzii Craib chloroplasts were sequenced, their assembly annotation and structural characterization were completed, and the genomes of the congeners Alangium chinense and Alangium alpinum were downloaded from NCBI for comparative analysis. Finally, the phylogenetic tree was constructed by selecting published species with close affinity to Alangium chinense family from NCBI. The results were as follows: Alangium chinense subsp. Pauciflorum, Alangium chinense subsp. Strigosum and Alangium kurzii Craib chloroplast genomes were 156 670, 156 672 and 156 871 bp in length, with a typical four-segment structure, all containing one long single copy region (LSC), one short single copy region (SSC) and two inverted repeat regions (IRa and IRb). All of them were annotated to 133 genes, including 88 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Three types of long repeat sequences and SSR loci, forward repeats, palindrome repeats and reverse repeats, were found in all chloroplast genomes. Comparative genomic analysis showed that there was diversity in the boundary transition regions of the five species, no rearrangements or inversions were found in the chloroplast genome, and there were significant differences in the coding regions of ndhA, ycf1, rpl16, ycf2, and petD genes, which provided new loci for molecular identification of Patagonia. In the phylogenetic analysis, Alangium kurzii var. kurzii clustered as a single species, and Alangium chinense subsp. Pauciflorum and Alangium chinense subsp. Strigosum clustered as a single species, with a support rate of 93 and close affinity, indicating that the phylogenetic tree can be used for the species identification. This paper can provide a basis for the taxonomic identification of Alangium, ensure the safety of clinical use of anise herbs and regulate the market of Alangium chinense herbs.

italic>Ardisia crispa (Thunb.) A. DC. is a traditional Miao medicinal herb with significant therapeutic effects in the treatment of sore throat, tonsillitis, edema of nephritis and bruising and rheumatism, etc. Ardisia crispa var. amplifolia and Ardisia crispa var. dielsii are varieties of A. crispa. A. crispa var. amplifolia and A. crispa var. dielsii are controversial in terms of species evolutionary relationships and taxonomic identification. In this study, we sequenced the whole genome sequences of A. crispa var. amplifolia and A. crispa var. dielsii chloroplasts using Illumina platform, assembled, annotated and characterized them, compared the structural features and degree of variation among chloroplast genomes using bioinformatics methods, and also downloaded constructing phylogenetic trees to analyze the phylogenetic relationships of chloroplasts in Primulaceae and Myrsinaceae using whole genome sequence information. The results showed that the complete chloroplast genome sequences of A. crispa var. amplifolia and A. crispa var. dielsii were 156 749 bp and 156 748 bp in length, with 132 genes annotated, including 87 protein-coding genes; the codon preference of A/U was greater than that of G/C; The differences in the coding regions of rps15 and rpoB genes in the comparative genome analysis can be used as loci for molecular identification of the two species; the differences in the coding regions of ycf1, ycf2, rpoC1, ycf3, petD and rpl16 genes in the chloroplast genome compared with those of the same genus can be used as loci for identification of the genus. In the phylogenetic results, A. crispa var. amplifolia and A. crispa var. dielsii were clustered together with 100% support, indicating that they are closely related. In this research, we analyzed the chloroplast genome structure and phylogenetic relationships of A. crispa var. amplifolia and A. crispa var. dielsii, providing an important theoretical basis for their molecular identification, genetic variation, breeding and phylogenetic analysis.

Quantification of cytotoxic T lymphocytes (CTL) is extremely important due to the pivotal role they play in controlling pathogen infection and anti-tumor actions. Previously used methods for detecting specific CTL are usually indirect. In recent years, tetramer technology has been developed to directly visualize antigen-specific CTL efficiently, and become the critical approach in studying T cell immune responses. A simplified procedure for preparing tetramers is reported here in this paper and a tetramer loaded with human cytomegalovirus (HCMV) peptide was successfully obtained using this procedure, which possessed binding activity with specific CTL. The heavy chain of HLA-A * 0201 gene was cloned by RT-PCR from HLA-A2+ donor. An expression vector, encoding the extracellular domain of HLA-A * 0201 heavy chain (A2) fused with a BirA substrate peptide (BSP) at its carboxyl terminus, was constructed by PCR with cloned A2 gene as the template. The A2 heavy chain was expressed in Escherichia coli mostly in the form of inclusion body and purified by washing inclusion body. The monomer of soluble A2 loaded with peptide was reconstructed by dilution from the heavy chain in the presence of light chain beta2-microglobulin and HLA-A2 restricted HCMV pp65(495-503) peptide (NLVPMVATV, NLV). Refolded A2-NLV monomer was biotinylated with a commercial BirA and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramer was then formed by mixing A2-NLV monomer with streptavidin-PE in a ratio of 4:0.8 leading to more than 85% multiplication as revealed by SDS-PADE under non-reducing conditions without boiling the sample. Flow cytometry analysis indicated that this tetramer could bind to specific CTL from HLA-A2+ donor. In conclusion, a simplified procedure is established to prepare HLA-A2 tetramer, which may not only facilitate the application of tetramer technology for studying specific T lymphocyte immune response but A2-NLV itself be applied clinically to monitor CMV-specific CTL in stem cell and organ transplantation.
Cloning, Molecular ; Cytomegalovirus ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; HLA-A Antigens ; biosynthesis ; genetics ; immunology ; HLA-A2 Antigen ; Humans ; Phosphoproteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; Viral Matrix Proteins ; biosynthesis ; genetics

OBJECTIVETo explore the cure effects of Jiangu Fufang on osteoporotic model induced by ovariectomy.

METHODRats were ovariectomized and administered drugs for 3 monthes. Bone mineral density and biomechanics properties, histomorphometric analysis and biochemical index such as calcium, phosphorus, alkaline phosphatase were detected.

RESULTJiangu Fufang could significantly increase bone density and biomechanics properties. The level of calcium, phosphorus and alkaline phosphatase were restored by Jiangu Fufang. Jiangu Fufang could significantly increase area of bone trabecula, thickness of cortical bone and bone trabecula.

CONCLUSIONJiangu Fufang could cure osteoporosis through increasing bone mineral density, improving bone biomechanics properties, and effecting bone metabolism.

Alkaline Phosphatase ; blood ; Animals ; Biomechanical Phenomena ; Bone Density ; drug effects ; Calcium ; blood ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Female ; Femur ; physiopathology ; Lumbar Vertebrae ; drug effects ; pathology ; physiopathology ; Osteoporosis ; blood ; drug therapy ; physiopathology ; Ovariectomy ; Phosphorus ; blood ; Phytotherapy ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene.

METHODSLentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay.

RESULTSThe MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%.

CONCLUSIONSThe lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Dogs ; Factor IX ; genetics ; metabolism ; Genetic Vectors ; Hemophilia B ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; Plasmids ; genetics ; Transfection

OBJECTIVETo study the effect of natural killer (NK)-cell on reconstitution of hematopoiesis and immunity in mouse allogeneic bone marrow transplantation (allo-BMT).

METHODSLethally irradiated BALB/c (H-2(d)) mice were transplanted with C57BL/6 (H-2(b)) bone marrow plus peripheral T cells and/or NK cells. Recipients CD34(+) cells and H-2K(b+), CD3(+) and CD19(+) cells were detected by flow cytometry, peripheral white blood cell (WBC) by auto-cytometry, and the survival rates, engraftment, hematopoietic and immune recovery were observed.

RESULTSIn the transplantation with NK cell infusion group, the survival rates, the WBC and CD34(+) cell counts, and the H-2(b+) and CD19(+) cells were significantly higher than that in without NK cell infusion group (P < 0.01). Twenty-eight days after transplantation, the CD3(+) cells in the NK cell infusion group [(33.69 +/- 3.36)%] were lower than that in without [(50.4 +/- 5.06)%] (P < 0.01), and there was no longer difference between these groups 60 days after transplantation (P > 0.05).

CONCLUSIONIn mouse allo-BMT, alloreactive NK cell enhances engraftment, promotes reconstitution of hematopoiesis and immunity and increases survival rates.

Animals ; Antigens, CD19 ; analysis ; Antigens, CD34 ; analysis ; Bone Marrow Transplantation ; immunology ; methods ; Cells, Cultured ; Female ; Flow Cytometry ; Graft Survival ; immunology ; Hematopoiesis ; immunology ; Killer Cells, Natural ; cytology ; immunology ; transplantation ; Lymphocyte Transfusion ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transplantation, Homologous

BACKGROUND: Few studies investigated serum uric acid levels in patients with acute ST-elevation myocardial infarction (STEMI). The study was to assess the clinical value of serum uric acid levels in patients with acute ST-elevation myocardial infarction (STEMI). METHODS: Totally 502 consecutive patients with STEMI were retrospectively studied from January 2005 to December 2010. The level of serum lipid, echocardiographic data and in-hospital major adverse cardiovascular events (MACE) in patients with hyperuricemia (n=119) were compared with those in patients without hyperuricemia (n=383). The relationship between the level of serum uric acid and the degree of diseased coronary artery was analyzed. All data were analyzed with SPSS version 17.0 software for Student's t test, the Chi-square test and Pearson's correlation coefficient analysis. RESULTS: Serum uric acid level was positively correlated with serum triglyceride level. Hyperlipidemia was more common in hyperuricemia patients than in non-hyperuricemia patients (43.7％ vs. 33.7％, P=0.047), and serum triglyceride level was significantly higher in hyperuricemia patients (2.11±1.24 vs. 1.78±1.38, P=0.014). But no significant association was observed between serum uric acid level and one or more diseased vessels (P>0.05). Left ventricular end-diastolic diameter (LVEDd) was larger in hyperuricemia patients than in non-hyperuricemia patients (53.52±6.19 vs. 52.18±4.89, P=0.041). The higher rate of left systolic dysfunction and diastolic dysfunction was discovered in hyperuricemia patients (36.4％ vs. 15.1％, P<0.001; 68.2％ vs. 55.8％, P=0.023). Also, hyperuricemia patients were more likely to have in-hospital MACE (P<0.05). CONCLUSIONS: Serum uric acid level is positively correlated with serum triglyceride level, but not with the severity of coronary artery disease. Hyperuricemia patients with STEMI tend to have a higher rate of left systolic dysfunction and diastolic dysfunction and more likely to have more in-hospital MACE.

Objective To investigate the level of homocysteine among healthy examination people and the possible related factors.Methods Retrospective analysis was performed to collect 1 259 results of healthy examination people from July to September in 2015,and 564 patients were confirmed to be hyperhomocysteinemia.The serum level of lipoids,glucose,uric acid and blood routine results were also collected.Results The incidence of hyperhomocysteinemia was 45.52％,and man has a higher rate than woman.The results of logistic regression showed positive results of UA (OR =1.006,95％ CI =1.005-1.008),HBG(OR =1.035,95％CI=1.026-1.045),and PLT (OR =0.996,95％ CI =0.993-0.998) in high hyperhomocysteinemia patients.Condusion High UA、HBG and low PLT levels are risk factors in hyperhomocysteinemia,and could be the important way for the early diagnoses of hyperhomocysteinemia.

Human beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC I tetramer. The present study aims to obtain recombinant human beta2m expressed in Escherichia coli (E. coli), for the purpose of preparing MHC class I tetramers. For cloning of human beta2m gene, a pair of specific primers was designed based on the published sequence of this gene and the cDNA of full coding region for beta2m precursor was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was subsequently cloned and its sequence was confirmed by DNA sequencing analysis (the sequence has been deposited in GenBank with accession number of AY187687). The prokaryotic expression vector containing a gene encoding mature beta2m was constructed by inserting the DNA fragment, which was generated by PCR reaction with the cloned beta2m gene as template, into an IPTG-inducible expression vector pET-3c plasmid. The first eight codons for N terminal amino acid residues of beta2m were optimized for its expression in E. coli. The complete sequence of beta2 m gene in the expression vector was verified by DNA sequencing analysis. High-yield expression of beta2m was achieved in E. coli transformed with the expression vector, and most of the recombinant beta2m existed in the inclusion body after IPTG induction. The inclusion body was washed extensively and beta2m in the inclusion body was solublized with 8 mol/L urea. The beta2m was refolded by dialysis and purified by ion-exchange chromatography (Q-Sepharose). Western blotting assay indicated that the polyclonal antibody against human native beta2m could react specifically with the recombinant protein. The purified protein appeared as a single band on both SDS-PAGE and Western blotting, indicating that it was chemical and antigenic pure. This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta2m which is identical to the native protein without any tags fused except for a methionine residue at the amino terminus. This provides the basis for the preparation of MHC tetramers.
Base Sequence ; Blotting, Western ; Cloning, Molecular ; Escherichia coli ; genetics ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; biosynthesis ; chemistry ; immunology ; beta 2-Microglobulin ; chemistry ; genetics ; immunology