siRNA-mediated inhibition of HBV replication and expression

AIM: RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, we investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. METHODS: pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR.RESULTS: Vector-based siRNA could potently reduce hepatitis 13 virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vectorbased siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. CONCLUSION: Our results indicate that RNA interference may be a potential tool to control HBV infection.

TGF-β1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-b 1 (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of α5β1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment. Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-β1-enhanced cell migration.

Downregulation of wild-type p53 protein by HER-2/neu mediated PI3K pathway activation in human breast cancer cells: its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy. Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused Gl-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.

Gene expression profiles in an hepatitis B virus transfected hepatoblastoma cell line and differentially regulated gene expression by interferon-α

AIM: To study interactions between hepatitis B virus (HBV) and interferon-a in liver- derived cells. METHODS. mRNAs were separately isolated from an HBV-transfected cell line (HepG2 2.2.15) and its parental cell line (HePG2) pre- and post-interferon-a (IFN-a) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis. RESULTS: Compared to HepG2 cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG2 2.2.15 cells prior to IFN-a treatment. After interferon-a treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG2 2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-a treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions. CONCLUSION: The up-regulated genes in HepG2 2.2.15 cell line suggested that under IFN-a treatment, these repressed cellular genes in HBV infected hepatooltes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.

Optimization of measurement distance of (109)~Cd K XRF system for obese subjects

The precision of results obtained from the ^109Cd K XRF in vivo measurement system of bone lead for obses subjects with high BMI( body mass index)was poor.The main factor affecting the precision was the distance between tibia and detector.Compared with the standard phantom,a large phantom was used to simulate the obese subject in the measurements at different distances to the detector.The counts of Compton scattering increased highly because of the tissue overlying and surrounding tibia of the obese subject.When the distance between leg and detector was too small,the instrument would produce the distorted X-ray spectra,so that the obtained data were inaccurate,In order to ensure good measuremtn precision and accuracy,the distance between leg and detector should be maintained at 25mm during the counting period.Meanwhile,the dead time displayed instantly on the instrument should be controlled to around 30%.

S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin β1

AIM:To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721.METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent,and cells were screened by G418.RESULTS: Overexpression of α5β1 or β1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21cip1 and p27kip1. The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved.When phosphorylation of PKB was solely blocked by wortmannin, p27kip1 protein level was increased. Moreover,S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of polyHEME, and this cell cycle pattern was similar to that of β1-7721 or α5β1-7721 cells.CONCLUSION: S-phase delay induced by overexpression of integrin β1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21cip1 and p27kip1 proteins, and may be involved in the unoccupied α5β1because of lack of its ligands.

Tumor suppressor gene PTEN affects anoikis via both PKB and FAK in human lung carcinoma cell lines

PTEN was identified in 1997 as a new tumor suppressor gene on human chromosome 10q23, deletions and mutations of this gene were associated with a variety of human cancers such as glioblastoma and prostate cancer. In this study Northern blot analysis was carried out and one major band at 2 kb region was observed in all 8 HLCC samples, consistent with previous reports. The result showed that the PTEN gene were expressed and its mRNA level similar in all cell lines tested. To determine whether the PTEN mRNA level reflects the parallel level of protein, the level of PTEN protein was examined by Western blot.PTEN protein level was high in H460 and detectable in A549, A4, A7, L1 cells, not detectable in 95C, 95D,

Study on Differences in Spermatogenic Suppression between Azoospermic and Oligozoospermic Responders Treated with Levonorgestrel Implant Plus Testosterone Undecanoate Injectable in Chinese Men

Objective To investigate possible causes resulting in the differences in the spermatogenesis suppression on individual treated with levonorgestrel (LNG) implants and testosterone undecanoate (TU) injectableMethods Totally 21 Chinese male volunteers were given treatment with LNG implants (four rods, 75 mg/rod) and intramuscular injection of TU (500 mg,bimonthly for 3 times). According to the effects of treatment, they were divided into two groups, namely, azoospermia group (group A) and oligozoospermia group (group O). Then seminal FSH, LH, T and estradiol (E2) were determined by immunoenzymetric assay, while seminal and serum dihydrotachysterol (DHT) and serum sex hormone binding globulin (SHBG) were by radioimmunoassay, and seminal transferrin (Tf) by scatter turbidimetry assay.Results Seminal FSH, LH and serum DHT, SHBG, FTI (T/SHBG ×100) levels were significantly lower in group A than in group O, while higher seminal concentrations ofE2 were observed in azoospermia group.Conclusion The differences in the spermatogenic suppression in Chinese men might be attributed to different rate of peripheral androgen metabolism, variations in serum SHBG levels, 5á-reductase activity and individual aromatase activity during LNG plus TU administration. In addition, seminal sex hormones might be more sensitive indexes to assess the extent of feedback inhibition on hypothalamus-pituitary-testis with exogenous testosterone plus progestogen in the efficacy hormone male contraceptive trials.

Expression of Human Chorionic Gonadotropin β(hCGβ) in Lactobacillus Casei

Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein from Lb. brevis and then cloned into down-stream of lactose-inducible promoter of an integrative plasmid, pIlac. After electroporation into Lb. casei CECT5276, PCR using the genomic DNA of the recombinant lactobacillus as template was performed to confirm whether the hCGβ gene had been integrated into the genome. Radioimmunoassay (RIA) was used to determine the level of hCGβ in the supernatant and the cell lysate. Results The hCGβ was integrated into the genome of Lb. casei CECT5276. The highest concentration of hCGβ in the culture supernatant amounted to 440 mIU/mL 21 h after lactose induction. About 2/3 of the abstract Objective proteins were excreted into the supernatant. Conclusion We have obtained stable and efficient hCGβ excretion in Lb. casei, which was inducible by lactose.

Improving Demand-oriented Quality Care in Family Planning-A Review of Practice and Experience in Family Planning Programme of Qianjiang, Hubei

With the mainstreaming being the demand from the people at reproductive age, we systematically analyzed the ideas and ways to implement quality care (QC) in family planning (FP) in Qianjiang, including advocating the conception of quality care, carrying out health education and counseling, strengthening capacity building of service system and reforming measurement of the evaluation and other aspects. The demand-oriented QC in FP has met personalized and verified demands from people of reproductive age satisfactorily, and kept the fertility rate at a lower level while uplifting satisfaction of the public. The demand-oriented QC in FP in Qianjiang county proved to be a successful and great worth practice.

Antinociception of ciproxifan in mice and its central mechanisms

AIM To investigate the effects of ciproxifan (CPF), an H3 receptor antagnist, on modulation of pain transmission in mice and its central mechanism. METHODS The antinociceptive effect of CPF was observed in three hyperalgesic models of mice (hot plate test, writhing test and formalin test). At the same time, α-fluoromethylhistidine (α-FMH), a specific inhibitor of histidine decarboxylase(HDC), was used to determine whether histamine participate in this process. After formalin test, the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) in brain, spinal and serum were assessed. Furthermore, the activation of neuronal nitric oxide synthase (nNOS) in brain and spinal cord was observed by immunohistology and Western blot in formalin test. RESULTS CPF produced antinociceptive effect inall of the three hyperalgesic models.

A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization.Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids.The mice with effective antibodies induced were used for preparation of mAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology. 2004;1(4):295-299.

Mutations of connexin43 in fetuses with congenital heart malformations

Background Gap junction channels formed by connexin43 (Cx43) protein are important in cardiac morphogenesis, and Cx43 gene is thought to be associated with congenital heart malformation (CHM). This study was undertaken to detect the mutations of Cx43 in fetuses with CHM.Methods Cx43 extron DNA was amplified by PCR from 16 fetuses with a variety of CHM. The PCR products were analyzed by SSCP and DNA sequencing. Thirty children who had no CHM were selected as controls. Results Eight homozygous mutations of Cx43 were observed in a fetus with double outlet right ventricule (DORV), five of the 8 mutations were missense mutations including Arg239Trp, Ser251Thr, Ala253Pro, Pro283Leu and Thr290Asn, and the remaining 3 were silent polymorphisms including Gly252Gly, Pro256Pro and Thr275Thr. No mutations were found in other fetuses and the control group.Conclusions Mutations of Cx43 may be associated with congenital conotruncal anomalies. PCR-SSCP is an effective method for screening the mutations of Cx43.

Detection of herpes simplex virus type 1 in rheumatic valvular tissue

Background Rheumatic heart disease (RHD) is the most important sequela of rheumatic fever (RF): evidence that streptococcal infection is aetiological is prominent, but sometimes contradictory. Acute HSV-1 infection in mouse leads to carditis and valvulitis whereas recurrent infection results in inflammatory granulomatous lesions that resemble Aschoff bodies. Cells containing HSV-1 inclusions or virus infected giant cells appear similar to Anitschkow cells or Aschoff cells respectively. We hypothesized that HSV-1 infection also may be involved in RHD. Methods Formalin-fixed, paraffin-embedded valvular tissue samples from 32 patients with RHD were investigated for evidence of HSV-1 infection. HSV-1 antigen was detected by immunohistochemistry, using HSV-1-specific monoclonal and polyclonal antibodies. HSV-1 glycoprotein D gene sequences were amplified by nPCR, using β-globin gene amplification in the same samples as internal control. Valvular tissue from 5 cases of sudden death and 3 cases died of neisseria meningitis without a history of valvular disease was used for comparison. HSV-1-infected lung tissue was used as positive control. Results HSV-1 antigens were detected in valvular tissues from 21 of 32 (65.6%) patients. Fifteen of these 21 (46.9% of cases), but no antigen-negative sample, were positive also for HSV DNA. Nucleotide sequence of PCR products was homologous to the targeted region of the HSV-1 glycoprotein D gene. HSV-1 antigen was present also in one case of sudden death but viral DNA was not found in any tissue sample from the comparison group. Results from reagent and positive controls were as anticipated.Conclusions This is the first study to show the presence of HSV-1 antigen and genomic DNA in valvular tissues from patients with RHD and provides evidence for an association of HSV-1 infection with some cases of rheumatic valvular disease.