OBJECTIVETo investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell.

METHODS(1) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4.1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)], blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 µg protein), CASK antibody precipitation group (100 µg protein), IgG antibody group (100 µg protein), Western blot group (20 µg protein). Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn), KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/L PBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group (transfected with pcDNA-Achn vector), and blank control group (treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test.

RESULTS(1) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10.777, 6.112, P values all below 0.05). The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3.1 group (with t value respectively 5.367, 6.053, 9.831, P values all below 0.05). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group (with t value respectively 5.481, 9.517, P values all below 0.05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [(15.6 ± 0.5)%] as compared with that in LPS induction group [(32.8 ± 2.6)%, t = 10.083, P < 0.05], and that in cotransfection group showed further inhibition [(7.0 ± 2.0)%, t = 9.827, P < 0.01]. (4) Apoptosis rate in Achn inhibition group [(45.6 ± 10.9)%] was higher than that in blank control group [(13.2 ± 4.3) %, t = 7.043, P < 0.05]; while that in Achn induction group [(5.3 ± 2.9)%] was lower than that in blank control group (t = 6.499, P < 0.05).

CONCLUSIONSAchn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.

Apoptosis ; Autoantigens ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Endothelial Cells ; cytology ; Guanylate Kinases ; metabolism ; Humans ; Ribonucleoproteins ; genetics ; metabolism ; Transfection

OBJECTIVETo study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein.

METHODSFour specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR.

RESULTSIn comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells.

CONCLUSIONThere is a correlation between La protein and HBV mRNA and the expression of HBV protein.

Autoantigens ; metabolism ; Cell Line, Tumor ; DNA, Viral ; Hepatitis B Surface Antigens ; Hepatitis B virus ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; RNA, Viral ; Ribonucleoproteins ; metabolism

MicroRNAs (miRNAs) recruit the RNA-induced silencing complex (RISC) to repress the translation of target mRNAs. While the 5' 7-methylguanosine cap of target mRNAs has been well known to be important for miRNA repression, the underlying mechanism is not clear. Here we show that TNRC6A interacts with eIF4E2, a homologue of eIF4E that can bind to the cap but cannot interact with eIF4G to initiate translation, to inhibit the translation of target mRNAs. Downregulation of eIF4E2 relieved miRNA repression of reporter expression. Moreover, eIF4E2 downregulation increased the protein levels of endogenous IMP1, PTEN and PDCD4, whose expression are repressed by endogenous miRNAs. We further provide evidence showing that miRNA enhances eIF4E2 association with the target mRNA. We propose that miRNAs recruit eIF4E2 to compete with eIF4E to repress mRNA translation.
Autoantigens ; metabolism ; Cell Line ; Eukaryotic Initiation Factor-4E ; metabolism ; Gene Silencing ; Humans ; MicroRNAs ; genetics ; Protein Transport ; RNA, Messenger ; biosynthesis ; genetics ; RNA-Binding Proteins ; metabolism

OBJECTIVETo screen the proteins interacting with FXR1P for functional investigation of FXR1P.

METHODSThe yeast strain AH109 transformed with the recombinant expression vector pGBKT7/FXR1 was mated with the yeast strain Y187 pretransformed with human fetal brain cDNA library. The positive clones were screened and identified by sequence analysis.

RESULTSThe recombinant expression vector pGBKT7/FXR1 was constructed successfully. Five proteins binding to FXR1P were screened from human fetal brain cDNA library using the yeast two-hybrid system, including CMAS, FTH1, GOLGA4, HSD17B1 and CSH1.

CONCLUSIONSThese results provide new clues for investigating the biological functions of FXR1P and the pathogenesis of Fragile X syndrome.

Autoantigens ; genetics ; metabolism ; Estradiol Dehydrogenases ; genetics ; metabolism ; Ferritins ; genetics ; metabolism ; Gene Library ; Humans ; Membrane Proteins ; genetics ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

Animals ; Autoantigens ; genetics ; metabolism ; Cell Line, Tumor ; Golgi Apparatus ; genetics ; metabolism ; Golgi Matrix Proteins ; Insulin-Secreting Cells ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proinsulin ; genetics ; metabolism ; Rats ; rab1 GTP-Binding Proteins ; genetics ; metabolism

Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.
Angiogenesis Inhibitors ; metabolism ; Autoantigens ; genetics ; metabolism ; Cell Proliferation ; Cells, Cultured ; Collagen Type IV ; genetics ; metabolism ; DNA, Complementary ; genetics ; Electroporation ; Endothelial Cells ; cytology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Umbilical Cord ; cytology

Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.
Animals ; Autoantigens ; metabolism ; Cells, Cultured ; Flow Cytometry ; Gene Knockdown Techniques ; Genetic Vectors ; Hematopoietic Stem Cells ; cytology ; Humans ; Lentivirus ; genetics ; Mice ; Plasmids ; Ribonucleoproteins ; metabolism ; Transfection

Animals ; Autoantigens ; genetics ; metabolism ; physiology ; Centromere Protein A ; Chromosomal Instability ; Chromosomal Proteins, Non-Histone ; genetics ; metabolism ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Kinetochores ; metabolism ; Mitosis ; physiology ; Rectal Neoplasms ; genetics ; metabolism ; pathology ; Spindle Apparatus ; metabolism

OBJECTIVETo express and identify soluble liver antigen (SLA) and cytochrome P-450 (CYP 2D6).

METHODSSLA cDNA and CYP 2D6 cDNA were obtained from human liver tissue poly (A)+RNA by RT-PCR. The cDNAs were inserted into fusion expression vector PQE-30 site of BamH I and Hind III. SLA and CYP 2D6 were identified by the SDS-PAGE and Western blot.

RESULTSSDS-PAGE analysis showed that there was a very strong stained band at about 47 kd and 50 kd, respectively. The products could specifically band to anti-SLA or anti-CYP 2D6 autoantibodies.

CONCLUSIONSThe clone and expression of SLA and CYP 2D6 provide useful substances for the diagnosis and research of pathogenesis on autoimmune hepatitis.

Autoantigens ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Hepatitis, Autoimmune ; genetics ; Humans ; Liver ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo investigate the mRNA expressions of RASSF1A, Galectin-3 and TPO in papillary thyroid carcinoma and some other thyroid benign lesions, and evaluate their diagnostic significance.

METHODSReverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of RASSF1A, galectin-3 and TPO in the samples from 73 cases, including 23 cases with papillary thyroid cancer, 16 with nodular goiter, 29 with thyroid adenoma and 5 with Hashimoto's disease.

RESULTSA statistically significant difference in the mRNA expression of RASSF1A, Galectin-3 and TPO was observed between papillary thyroid carcinoma and follicular benign lesions (P<0.05). However, there was no significant difference among various kinds of benign lesions (P>0.05). A negative correlation of the expression of RASSF1A and Galectin-3 mRNA was found between thyroid benign lesions and malignant ones (P = 0.000). While the mRNA expression of RASSF1A and TPO was positively correlated between benign and malignant lesions (P = 0.028).

CONCLUSIONLoss of expression of RASSF1A and TPO mRNA but high expression of Galectin-3 mRNA in papillary thyroid carcinoma are common. Therefore, the products of these three genes may be closely related to the development of thyroid papillary carcinoma, and may be used as useful markers in differential diagnosis of papillary thyroid carcinoma from the benign lesions. The results are more reliable if this detection method is used in combination with other techniques.

Adolescent ; Adult ; Aged ; Autoantigens ; genetics ; metabolism ; Biomarkers, Tumor ; metabolism ; Carcinoma, Papillary ; genetics ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Galectin 3 ; genetics ; metabolism ; Goiter, Nodular ; genetics ; metabolism ; pathology ; Hashimoto Disease ; genetics ; metabolism ; pathology ; Humans ; Iodide Peroxidase ; genetics ; metabolism ; Iron-Binding Proteins ; genetics ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; metabolism ; Thyroid Neoplasms ; genetics ; metabolism ; pathology ; Tumor Suppressor Proteins ; genetics ; metabolism ; Young Adult