Cyanovirin-N (CVN) is an 11 kDa anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and irreversibly inactivates diverse HIV strains. A fusion gene consisting of cvn, sumo and 6xHis tag was synthesized by PCR according to the codon bias of Escherichia coli. The fusion protein is expressed in the cytoplasm of E. coli in a soluble form and up to 28% of the total protein. The recombinant CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage. Bioactivity assay demonstrated that SUMO-CVN and CVN bound to gp120 with nanomolar concentration. In addition, CVN showed potent anti-HSV-1 and anti-HIV-1 activities in in vitro cellular assays. Therefore, the 6xHis SUMO fusion expression and purification system provides a better approach for large scale production of CVN for further microbicide development.
Antiviral Agents ; isolation & purification ; metabolism ; pharmacology ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; HIV-1 ; drug effects ; Herpesvirus 1, Human ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology

OBJECTIVETo investigate the gene expression of MAPEG in the cortex of concanavalin A (Con A)-induced mouse immune inflammatory model and the effect of cyclosporine A (Cs A).

METHODSMale Balb/c mouse immune inflammation model was developed by intravenous injection of Con A (20 mg/kg). Cs A (150 mg/kg) was intravenously infected prior to Con A administration. The MAPEG expressions were determined by RT-PCR.

RESULTmGST1, mGST3, LTC(4)S, FLAP and mPGES-1 were detected by RT-PCR but not mGST2. Eight hours after Con A treatment, mGST1 level was up-regulated to 1.2 approximately 1.5 folds of control with or without Cs A treatment. mGST3ìLTC(4)S, FLAP and mPGES-1 mRNA levels were not influenced by Con A administration.

CONCLUSIONImmune mechanism may be not involved in mGST1 up-regulation in this model and Con A does not alter arachidonic acid metabolism in cortex.

5-Lipoxygenase-Activating Proteins ; Animals ; Brain ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Concanavalin A ; toxicity ; Cyclosporine ; pharmacology ; Eicosanoids ; metabolism ; Glutathione ; metabolism ; Glutathione Transferase ; genetics ; metabolism ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Prostaglandin-E Synthases

OBJECTIVETo construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein.

METHODSGenes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method.

RESULTS(1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization.

CONCLUSIONpBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.

Antimicrobial Cationic Peptides ; Blood Bactericidal Activity ; drug effects ; Blood Proteins ; biosynthesis ; genetics ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Cell Adhesion Molecules ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Membrane Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

To explore the mechanism for the role of autophagy in endometriosis, and to provide a theoretical basis for prevention and treatment of endometriosis.
 Methods: The endometrial CRL-7566 cells were treated with ATG5 siRNA, autophagic activator rapamycin and autophagic inhibitor 3-MA, respectively. The cell proliferation and invasion were detected by clonal formation, cell growth curve and MTT assay. The clinical specimens of endometriosis were collected from 20 cases. The expression of autophagy marker LC3II and autophagy substrate protein P62 were detected.
 Results: Rapamycin inhibited the proliferation and clonal formation of CRL-7566 cells, while autophagy inhibitor 3-MA and ATG5 siRNA showed opposite effect. Moreover, rapamycin inhibited filopodia growth in endometriosis, whereas overexpression of filopodia-relevant protein fascin-1 inhibited the decrease in invasiveness caused by rapamycin. In clinical samples, we also found a significant decrease of LC3II while an increase in P62 compared with the control group.
 Conclusion: Autophagy inhibition may contribute to an increase in endometrial cell proliferation and invasiveness. Autophagy activation could be a potential strategy for endometriosis therapy.
Autophagy ; drug effects ; genetics ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Endometriosis ; physiopathology ; Endometrium ; cytology ; Female ; Gene Expression Regulation ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Microtubule-Associated Proteins ; genetics ; RNA-Binding Proteins ; genetics ; Sirolimus ; pharmacology

Novel organic cation transporter-2 (OCTN2), a member of the organic cation transporter family, may transport carnitine and multiple organic cationic drugs. Thus OCTN2 possesses substantial roles in physiology and pharmacology. A number of researches have proven that many factors can regulate the expression and/or function of OCTN2 via different pathways, and then may affect the homeostasis and disposition of drugs. This paper reviews recent progresses in this field.
Animals ; Biological Transport ; Carnitine ; metabolism ; Carrier Proteins ; physiology ; Clofibrate ; pharmacology ; Colitis ; metabolism ; Homeostasis ; drug effects ; Humans ; Mutation ; Organic Cation Transport Proteins ; genetics ; metabolism ; physiology ; PPAR alpha ; agonists ; RNA, Messenger ; metabolism ; Solute Carrier Family 22 Member 5

OBJECTIVETo study the effect of silencing pyruvate kinase M2 (PKM2) on gambogic acid (GA)-induced apoptosis of human prostate cancer PC3 cells.

METHODSThree specific PKM2 siRNAs and one negative control siRNA (si-NC) were transfected into PC3 cells. The silencing effect of PKM2 siRNAs was determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, and the effects of PKM2 siRNA on the vitality and apoptosis of GA-stimulated PC3 cells detected by MTT and AO/EB double staining, respectively. The mRNA and protein levels of c-myc and cyclin D1 were analyzed by qRT-PCR and Western blot, respectively.

RESULTSAll the 3 PKM2 siRNAs effectively reduced the mRNA and protein expressions of PKM2, and PKM2 siRNA-1 exhibited the strongest silencing effect. At 24 h after transfection, the expression levels of PKM2 mRNA and protein were reduced by 70% and 85%, respectively (P < 0.05). Twenty-four hours of treatment with GA (0.5 micromol/L) following transfection with PKM2 siRNA-1 inhibited the vitality of the PC3 cells by 68%, increased their apoptosis, and significantly down-regulated the mRNA and protein levels of c-myc (50% and 35%) and cyclin D1 (60% and 20%) (P < 0.05).

CONCLUSIONInhibition of PKM2 sensitized PC3 cells to GA-induced apoptosis, suggesting that PKM2 may be a potential therapeutic target for sensitizing human prostate cancer to GA.

Apoptosis ; drug effects ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; Thyroid Hormones ; genetics ; metabolism ; Xanthones ; pharmacology

OBJECTIVESTo investigate the expression status of IER3IP1 gene during matrine induced K562 cell differentiation, and to figure out the function of IER3IP1 gene in K562 cell line.

METHODSTrypan-blue staining was used to analyze the growth inhibitory effect of matrine on K562 cells. Semi-quantitative RT-PCR was employed to investigate the expression status of IER3IP1 gene treated with different time and dosage of matrine. The alteration of cellular morphology, cellular proliferation and ultra-microstructure were observed and the cell cycle was detected on the recombinant IER3IP1 gene eukaryotic expression vector eYFP-IER3IP1 plasmid transfected K562 cells (K562/eYFP-IER3IPl).

RESULTSMatrine inhibited the growth of K562 cells and reduced the expression of IER3IP1 gene. The expression level of IER3IP1 gene was transiently increased to three-to-four times in a dose-dependent manner after treated with matrine for 2 - 3 hours. Then, in 6-48 hours it maintained at a low level as compared with the control group. The proliferation rate of the K562/eYFP-IER3IP1 cells significantly slowed down with more cells blocked in G0-G1 phase (P < 0.05). The number of erythroid blast cells began to increase after 24 hours of matrine treatment. At the same time, differentiated erythroid cells could be observed.

CONCLUSIONSMatrine can inhibit the growth of K562 cells, and transiently increase the expression level of IER3IP1 gene in a dose-dependent manner. The sensitivity of K562 cells to matrine maybe increased after being transfected by the eYFP-IER3IP1 plasmid, indicating a possible involvement of the IER3IP1 gene in the early response of the cells to matrine and its possible role in the erythroid cell differentiation.

Alkaloids ; pharmacology ; Carrier Proteins ; genetics ; metabolism ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; Membrane Proteins ; genetics ; metabolism ; Quinolizines ; pharmacology

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.
Adaptor Proteins, Signal Transducing ; Carbachol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Chemokine CCL2 ; biosynthesis ; genetics ; Chemokines ; biosynthesis ; genetics ; Humans ; NF-kappa B ; biosynthesis ; genetics ; Pancreas, Exocrine ; metabolism ; Pancreatitis ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Muscarinic M3 ; agonists ; physiology ; Signal Transduction

OBJECTIVETo investigate the effects of all-trans-retinoic acid(ATRA) and polyphenon-100 (PP) on genetic instability of human tumor cells via their role in alteration of microsatellite sequence(MS) and the expression of mismatch repair gene hMLH(1) and hMSH(2) in RER(+) (replication error) cells.

METHODSRER(+) colon cancer cell line was used as a host for lipofection with pCMV-CAR in which a foreign (CA)(14) repeat was inserted in the coding sequence of LacZ reporter gene, resulting in misreading LacZ frame. Any mutation which made the base number of (CA)(14) tract to be 3-fold resumed normal reading frame of LacZ, and thus led to expression of beta-galactosidase. Variable expression of LacZ in the transfectant cells resulting from RATA or PP treatment was measured by OD reading at lambda 620 after X-gal staining. Expression of mismatch repair genes of hMLH(1) and hMSH(2) was examined at mRNA level by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSATRA at 1 mu mol/L, 0.1 u mol/L and PP at 3 mu g/ml had no significant inhibitory effect on cell proliferation. After being treated with ATRA or PP for 1 week, the blue cells of RKO transfectant clones were significantly reduced, and this meant the mutation of exogenous (CA)(14) in RKO cells were inhibited. But no expression of hMLH(1) and hMSH(2) was observed.

CONCLUSIONThe above data showed both ATRA and PP had inhibitory effects on MS instability of cancer and thus demonstrated directly their beneficial role in stabilization of genomic DNA. However, the present authors have not observed any expression of hMLH(1) and hMSH(2) in RKO cells treated with ATRA or PP.

Adaptor Proteins, Signal Transducing ; Carrier Proteins ; Catechin ; pharmacology ; Cell Division ; drug effects ; Colonic Neoplasms ; genetics ; pathology ; DNA-Binding Proteins ; Gene Expression ; drug effects ; Humans ; Lac Operon ; genetics ; Microsatellite Repeats ; genetics ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Mutation ; Neoplasm Proteins ; genetics ; Nuclear Proteins ; Plasmids ; genetics ; Proto-Oncogene Proteins ; genetics ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Transfection ; Tretinoin ; pharmacology ; Tumor Cells, Cultured ; drug effects

Fas transduces apoptotic signals upon cross-linking with the Fas ligand (FasL), which is experimentally replaced by agonistic anti-Fas monoclonal antibodies (mAb). Of eight human malignant hematopoietic cell lines (HL-60, KG-1, THP-1, K562, U937, Jurkat, IM-9, RPMI-8226) examined by flow cytometric analysis, all, except K562, were found to be positive for surface Fas antigen. However, despite surface Fas expression, the agonistic anti-Fas mAb (7C11) induced apoptosis in only three of seven Fas-expressing cell lines (KG-1, Jurkat and IM-9). This Fas-resistance did not correlated with high levels of mRNA either for DcR3, a decoy receptor for FasL, or for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not show consistent differences in the expression of Bcl-2 and Bax between Fas-sensitive and Fas-resistant cell lines examined. These findings indicated that the presence or absence of mRNA expression of DcR3, FAP-1, Bcl-2 and Bax did not always correlate with relative sensitivity to Fas-mediated apoptosis. Treatment of cells with cycloheximide converted the phenotype of resistant cell lines from Fas-resistant to Fas-sensitive, and enhanced the sensitivity of Fas-sensitive cell lines. These results suggest that the Fas-resistance is dependent on the presence of labile proteins that determine resistance to Fas-mediated apoptosis and the apoptotic machinery is already in place in Fas-resistant cell lines.
Antigens, CD95/*metabolism ; Apoptosis/drug effects/*genetics ; Carrier Proteins/biosynthesis/genetics ; Comparative Study ; Cycloheximide/pharmacology ; Gene Expression Regulation, Neoplastic ; Hematologic Neoplasms/*genetics/metabolism ; Human ; Membrane Glycoproteins/*metabolism ; Protein Synthesis Inhibitors/pharmacology ; Protein-Tyrosine-Phosphatase/biosynthesis/genetics ; Proto-Oncogene Proteins/biosynthesis/genetics ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/genetics ; Receptors, Cell Surface/biosynthesis/genetics ; Signal Transduction ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured