Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.
Amino Acids ; Catalytic Domain ; DNA, Complementary ; Echinostoma* ; Endoribonucleases ; Escherichia coli ; Intestine, Small ; Oligonucleotides, Antisense ; Parasites ; Ribonuclease H* ; Ribonucleases* ; RNA ; Trematoda

Mammals have 3 pairs of major salivary glands i.e., the parotid, submandibular, and sublingual glands. Saliva secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and ductal cells. Primary saliva is secreted by acinar cells and modified during ductal flow. Recently, of the murine 35 bitter taste receptors, Tas2r108 was expressed at highest levels in the submandibular gland by qPCR. Further, Tas2r108-transfected cells respond to a range of bitter compounds, such as denatonium, quinine, colchicine, diphenidol, caffeine and dapson. The objective of the present study was to characterize the expression of Tas2r108 mRNA in acinar and/or ductal cells of the submandibular gland using in situ hybridization (ISH). Male 42-60 days old DBA2 mice were used in the study. Messenger RNAs were extracted from the submandibular gland for generating digoxigenin (DIG) labeled-cRNA probes. These probes were transcribed in anti-sense and sense orientation using T7 RNA polymerase. Dot blot hybridization was performed using DIG labeled-cRNA probes, in order to estimate integrity and optimal diluting concentration of these probes. Subsequently, ISH was performed on murine submandibular gland to detect Tas2r108 mRNA. Dot blot hybridization data demonstrated that Tas2r108 DIG labeled-cRNA anti-sense probes specifically detected Tas2r108 cDNA. ISH results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular gland, whereas no staining was visible in sense controls. Interestingly, the Tas2r108 expression levels were higher in acinar than ductal cells. These results suggested that Tas2r108 might be more associated with primary saliva secretion than with ductal modification of saliva composition.
Acinar Cells ; Animals ; Antisense Elements (Genetics) ; Caffeine ; Colchicine ; Digoxigenin ; DNA, Complementary ; DNA-Directed RNA Polymerases ; Humans ; In Situ Hybridization* ; Male ; Mammals ; Mice ; Quinine ; RNA, Messenger ; Saliva ; Salivary Glands ; Sublingual Gland ; Submandibular Gland* ; Taste Perception

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.
DNA Primers ; Down-Regulation ; Gene Expression ; Intramolecular Transferases ; genetics ; metabolism ; Plasmids ; Polymerase Chain Reaction ; RNA, Antisense ; Saccharomyces cerevisiae ; enzymology ; genetics ; Squalene ; analogs & derivatives ; metabolism ; Transformation, Genetic

OBJECTIVE: To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. METHOD: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. RESULT: Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phβApr-neol cells (P < 0.01). CONCLUSION These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.
Animals ; DNA, Antisense ; therapeutic use ; DNA, Complementary ; Heparan Sulfate Proteoglycans ; genetics ; Heterografts ; Humans ; Laryngeal Neoplasms ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

This paper is aimed to investigate the inhibitory effects of hepatitis B virus (HBV) preC and C genes-specific antisense locked nucleic acid (LNA) on HBV replication and expression in transgenic mice. The antisense LNA, which was complementary to the preC and C gene region of HBV, was designed, synthesized, and injected into transgenic mice via the tail vein. Serum HBV DNA was tested with real-time PCR, and Serum HBsAg was tested with time-resolved fluorescence immune assay (TRFIA). Then the expression of HBcAg in the liver was detected with immuneohistochemistry. Serum ALB, ALT, BUN and CRea were measured with an antomatic biochemicall analyzer. It was found that 5 days after LNA injection, serum HBV DNA levels in the dual-target group were reduced by 53.72%, and serum HBsAg levels were decreased by 71.57%. These values were significantly higher than those in the control groups (P<0.05) and the expression levels of HBcAg in the liver were significantly lower than those in the control groups (P<0.05). The result also showed that there were no significant differences discovered in serum ALB, ALT, BUN and CR between the experiment groups and the control groups. The present study provides that antisense LNA targeting to both preC and C genes has shown strong inhibition on HBV replication and expression in transgenic mice, and stronger than target at single gene site.
Animals ; Antiviral Agents ; pharmacology ; DNA, Viral ; blood ; Female ; Gene Targeting ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Liposomes ; Male ; Mice ; Mice, Transgenic ; Oligonucleotides ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; Virus Replication ; drug effects

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; Humans ; Oligonucleotides, Antisense ; pharmacology

OBJECTIVE: To investigate the changes of mitochondrion by transferring antisense oligodeoxynucleotide Stat3 into laryngeal carcinoma Hep-2 cell, for elucidating the mechanism of laryngeal carcinoma Hep-2 cell apoptosis, for developing more effective treatment for laryngeal cancer. METHOD: The designed Stat3 ASODN was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Mitochondrion membrane potential, VDAC and Cyt-c were detected for determining the changes of mitochondrion. RESULT: MMP was fell, Cyt-c and VDAC were increased with the heighten concentration of ASODN. CONCLUSION Mitochondria approach play an important role in the apoptosis mechanism of human Hep-2 cell by Stat3. This research elucidated the regulating mechanism of Hep-2 cell proliferation by Stat3, provided a new research focus for clinical therapy.
Apoptosis ; Cell Proliferation ; Cytochromes c ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; STAT3 Transcription Factor ; genetics ; Transfection ; Voltage-Dependent Anion Channels ; metabolism

In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P < 0.05). After Lovo cells treated with apollon ASODN for 48 hours, the expression of the apollon mRNA level was suppressed significantly. And a marked concentration-dependent decline of cell proliferation and clone forming, increasing of cell apoptosis levels were observed. The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry. Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation. Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon can suppress the expression of apollon mRNA, and inhibit the proliferation, induce apoptosis, arrest cell cycle at S phase of colorectal cancer Lovo cells in vitro and enhance the chemo-sensitivity to 5-FU, DDP and EPI.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Colonic Neoplasms ; metabolism ; pathology ; Epirubicin ; pharmacology ; Fluorouracil ; pharmacology ; Gene Knockdown Techniques ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Inhibitory Concentration 50 ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; metabolism ; Sensitivity and Specificity ; Transfection

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Female ; Ferric Compounds ; chemistry ; Genes, erbB-2 ; genetics ; Humans ; Magnetics ; Microscopy, Atomic Force ; methods ; Molecular Probe Techniques ; Nucleic Acid Probes ; chemistry ; genetics ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; ultrastructure ; RNA, Messenger ; genetics ; metabolism

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.
Adenocarcinoma ; pathology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cyclin D1 ; genetics ; DNA, Antisense ; pharmacology ; Genetic Vectors ; Humans ; Lung Neoplasms ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Recombination, Genetic ; Retinoblastoma Protein ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism