Peptide nucleic acid(PNA) is a kind of artificial DNA mimic. PNA hybridizes with DNA or RNA by means of Watson-Crick's base-pairs complementary with high stability, affinity and selectivity. PNA not only regulates. DNA replication, but also adjusts DNA transcription (or reverse transcription) and translation. Many applications have been explored as a new kind of molecular biological tool and a gene-targeting strategy.
Gene Expression Regulation ; drug effects ; Peptide Nucleic Acids ; pharmacology ; Protein Biosynthesis ; drug effects ; Transcription, Genetic ; drug effects

OBJECTIVETo study the inhibitory effect of Jinye Baidu Preparation (JBP), a Chinese medicinal preparation, on human cytomegalovirus protein kinase pu197 and to explore its molecular mechanism in treating human cytomegalovirus (HCMV) infection.

METHODSExpression of the HCMV pu197mRNA in infected cells was measured by semi-quantitative RT-PCR before and after intervention of JBP or Ganciclovir (GCV), and effect of the two medicines on the proliferation activity of the infected cells was observed by MTT.

RESULTSBoth JBP and GCV showed obvious inhibitory action on HCMV pu197mRNA. They could significantly enhance the proliferation activity of the cells 72 hours after HCMV infection.

CONCLUSIONJBP could inhibit the gene expression and duplication of HCMV by inhibiting the gene expression of HCMV protein kinase pu197 to enhance the proliferation activity of the infected cells so as to achieve its anti-virus action.

Antiviral Agents ; pharmacology ; Cytomegalovirus ; drug effects ; enzymology ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Embryo, Mammalian ; Fibroblasts ; cytology ; virology ; Humans ; Lung ; cytology ; Protein Kinases ; biosynthesis ; drug effects ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Viral Proteins ; biosynthesis ; drug effects ; genetics ; Virus Replication ; drug effects

OBJECTIVETo evaluate the influence of minocycline on the proliferation and biosynthesis of human periodontal ligament cells (HPDLCs) in vitro.

METHODSVarious concentrations of minocycline (1, 5, 20, 100, 500, 2 500 mg/L) were added to the medium of cultured HPDLCs respectively. After co-incubated for 2 days, cell morphology was observed under reverse microscope, cell proliferation activity was assayed using MTT, the total amount of protein was detected with Coumassie Bright Blue method and DNA synthesis was measured by (3)H-TdR.

RESULTSThe presence of minocycline not exceeding 500 mg/L in the medium resulted in no morphological change of HPDLCs. Moreover, at a concentration range of 5 to 100 mg/L, minocycline significantly enhanced the proliferative activity and biosynthesis of HPDLCs (P < 0.01). However, higher concentration (2 500 mg/L) not only changed cell morphology under microscope, but also significantly inhibited cellular activity.

CONCLUSIONSThe results suggest that proper doses of minocycline could promote biobehavior of HPDLCs, while higher concentrations of minocycline had cytotoxic effect which may intervene affect tissue repair and regeneration.

Cell Division ; drug effects ; Cells, Cultured ; DNA ; biosynthesis ; Dose-Response Relationship, Drug ; Humans ; Minocycline ; pharmacology ; Periodontal Ligament ; cytology ; drug effects ; metabolism ; Protein Biosynthesis

OBJECTIVE: To investigate the effects of high dose vitamin C (VC) on proliferation of breast cancer cells and to explore its mechanisms. METHODS: Human breast cancer cells Bcap37 and MDA-MB-453 were treated with VC at low dose (0.01 mmol/L), medium dose (0.10 mmol/L) and high dose (2.00 mmol/L). Cell proliferation was determined with CCK-8 assay, protein expression was evaluated by Western blot, and the secretion of lactic acid in tumor cells was detected by colorimetric method. Bcap37 cells were inoculated in nude mice, and tumor baring nude mice were intraperitoneally injected with high VC(4 g/kg, VC group, =5)or normal saline (control group, =5) for 24 d. Tumor weight and body weight were calculated. RESULTS: experiments demonstrated that high dose VC significantly inhibited cell proliferation in Bcap37 and MDA-MB-453 cells (all <0.01); the expressions of Glut1 and mTOR signaling pathway-related proteins were decreased (all <0.05); and the secretion of lactic acid was also markedly reduced (all <0.05). experiment showed that the tumor weight was decreased in mice treated with high-dose VC as compared with control group (<0.05), but no difference in body weights between two groups was observed. CONCLUSIONS High dose VC may inhibit proliferation of breast cancer cells both and through reducing glycolysis and protein synthesis.
Animals ; Ascorbic Acid ; pharmacology ; Breast Neoplasms ; drug therapy ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Glycolysis ; drug effects ; Humans ; Mice ; Mice, Nude ; Protein Biosynthesis ; drug effects

OBJECTIVETo investigate the regulatory effect of interleukin-10 (IL10) on the activation of hepatic stellate cells (HSC) through platelet derived growth factor (PDGF) and mitogen-activated protein kinase (MAPK) pathways.

METHODSHSC were divided randomly into 4 groups. Group 1 served as a control. HSC were incubated with 1 ng/ml, 5 ng/ml, and 25 ng/ml IL-10 in groups 2, 3 and 4. RT-PCR and western blot were used to detect the expression of PDGF and MAPK protein ERK and p38 and alpha-SMA.

RESULTSCompared with the control group, expressions of ERK, p38 and alpha-SMA of groups 2, 3 and 4 were significantly lower (F values were 240.47, 21.39, 28.86 respectively. IL-10 inhibited PDGF and MAPK protein ERK and p38 and alpha-SMA expression in a dose-dependent way.

CONCLUSIONIL-10 inhibits activation of HSC through the PDGF/MAPK pathway.

Animals ; Cell Line ; Cell Proliferation ; drug effects ; Hepatocytes ; cytology ; drug effects ; Interleukin-10 ; pharmacology ; Mitogen-Activated Protein Kinases ; biosynthesis ; Platelet-Derived Growth Factor ; biosynthesis ; Rats ; Signal Transduction

OBJECTIVETo explore the mechanism of reactive oxygen species (ROS) in manganese chloride (MnCl(2))-induced apoptosis in PC12 cells.

METHODSThe model that MnCl(2) induced apoptosis in PC12 cells was established. The apoptotic effect of MnCl(2) on PC12 cells was analyzed with the MTT, the flow cytometry and the DNA fragmentation. The production of ROS and ATP in MnCl(2)-induced apoptosis of PC12 cells was examined. The influence of MnCl(2) on the expression of bcl-xl, bax and the activity of Caspase 3 was also analyzed.

RESULTSMnCl(2) triggered PC12 cells apoptosis in a dose-and time-dependent manner (P < 0.01). The rate of apoptosis was significantly increased (P < 0.01) when MnCl(2) of 2 mmol/L induced PC12 cells for 36 hours. The production of ROS was increased (P < 0.001) and the quantity of ATP was decreased (P < 0.01) in PC12 cells with the same inducement of MnCl(2). The expression of bcl-xl was inhibited and the bax was activated in this process (P < 0.01). Caspase 3 was also activated (P < 0.01).

CONCLUSIONMnCl(2) induces apoptosis of PC12 cells, which is related to the increase of ROS, the inhibition of the mitochondria and the activation of Caspase 3.

Adenosine Triphosphate ; biosynthesis ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Chlorides ; toxicity ; DNA Fragmentation ; drug effects ; Manganese Compounds ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

The intracellular mechanism by which interferon-gamma induces the expression of class II major histocompatibility complex (MHC) antigen in nonlymphoid cells is not clear. The effect of recombinant rat interferon-gamma (IFN-gamma), and cycloheximide on the expression of class II MHC gene was studied using the techniques of immunocytochemical staining and northern blot analysis. IFN-gamma induced de novo transcription of class II MHC gene and class II MHC antigen expression on the cell surface. Cycloheximide did not inhibit IFN-gamma-induced class II MHC antigen expression in a dose-dependent manner indicating translational blockade. These results suggest that IFN-gamma induces class II MHC antigen expression via de novo transcription of class II MHC gene leading to synthesis of new class II MHC molecule.
Animals ; Cell Line ; Cycloheximide/pharmacology ; Gene Expression Regulation/drug effects ; *Genes, MHC Class II ; Interferon-gamma/*pharmacology ; Protein Biosynthesis/drug effects ; Rats ; Transcription, Genetic/*drug effects

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.
Adenine ; administration & dosage ; analogs & derivatives ; Apoptosis ; drug effects ; Autophagy ; drug effects ; genetics ; CDC2 Protein Kinase ; Cell Cycle ; drug effects ; genetics ; Cell Division ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin B ; biosynthesis ; Cyclin-Dependent Kinases ; Gene Expression Regulation ; drug effects ; Humans ; Membrane Proteins ; biosynthesis ; Microtubule-Associated Proteins ; biosynthesis ; Mitochondrial Dynamics ; drug effects ; genetics ; Mitochondrial Proteins ; biosynthesis ; Neuroblastoma ; Signal Transduction ; drug effects

OBJECTIVETo investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.

METHODSROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.

RESULTS20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.

CONCLUSIONSSodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; biosynthesis ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

OBJECTIVESTo construct yeast expression plasmid of rat augmenter of liver regeneration (rALR), express it in GS115, and examine its bioactivity in vitro.

METHODSWith PCR and genetic recombination techniques, the gene fragment of rALR was amplified from recombinant plasmid pcDNA3.1-rALR, and the recombinant plasmid pPIC9K-rALR was constructed. The recombinant plasmid pPIC9K-rALR was transducted into GS115 by electroporation after identified with endonuclease digestion and PCR amplification. The target protein was expressed by GS115 under the induction of 0.5% methanol. The recombinant rALR (rrALR) was purified with ultrafiltration after demonstrated by 15% SDS-PAGE and western blot. The effects of rrALR on the proliferation of QGY, HepG2 cells and primary rat hepatocytes in vitro were evaluated by 3H-TdR intake method.

RESULTSRecombinant plasmid pPIC9K-rALR was identified by restriction digestion and PCR methods. The rrALR as a secretive protein was successfully expressed in GS115. Its molecular weight, 1.5x10(4), was in correspondence with theoretic value. The rrALR could bind with the polyclonal antibody against human ALR in western blot, which demonstrated that there was cross-antigenicity between human and rat ALR. The high qualitative rrALR was obtained through ultrafiltration. Among the experimental concentrations, the rrALR could stimulate the proliferations of QGY and HepG2 cells in a dose-dependent manner in vitro, but had little effect on the proliferation of primary rat hepatocytes in vitro.

CONCLUSIONSThe rrALR as a secretive protein is expressed in GS115 efficiently. The rrALR can stimulate the proliferations of QGY and HepG2 cells in a dose-dependent manner in vitro, but had no effect on primary rat hepatocytes, which demonstrates that there exists different receptors on the normal hepatocytes and hepatocarcinoma cells. There are cross-bioactivity and cross-antigenicity between human and rat ALR.

Animals ; Blotting, Western ; Dose-Response Relationship, Drug ; Hepatocytes ; drug effects ; Pichia ; genetics ; Polymerase Chain Reaction ; Protein Biosynthesis ; Proteins ; isolation & purification ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology