OBJECTIVETo construct a reverse transcriptase-polymerase chain reaction (RT-PCR)approach that can improve the specificity of primers while dropping down the nonspecific amplification.

METHODSIn the recent study we reported a new RT-PCR assay which improved markedly the specificity. However its efficiency of regressing nonspecific amplification remains to be accurately checked and further documented. In primer design, we looked over again some sequences that showed differences at 5' or 3' ends between human CAV1 and mouse Cav1 genes. cDNAs and the diluted plasmids which harbored the sequence of human CAV1 or mouse Cav1 gene were chosen as the templates. The ordinary PCR compared with one, of which primers modified by phosphorothioate and combined with proofreading polymerase, for their efficiencies of nonspecific amplification inhibited.

RESULTSTaq DNA polymerase without proofreading activity could efficiently catalyze the extension of primers with a single or multiple mismatched base pairs at the 3' terminus, but the kind of primer extension can be effectively blocked by phosphorothioate modified primers combined with proofreading polymerase. Compared with ordinary PCR reaction, this new PCR method can effectively regress the primer mismatched amplification of 50 ng DNA almost equaling to 2 x 10(4) unmatched template copies in a final volume of 50 microL.

CONCLUSIONCompared with the first generation of polymerases with or without proofreading activities mediating RT-PCR reaction, the introduction of nuclease-resistant 3' modified primers (3' phosphorothioate primer extension) can offer more simplicity, accuracy, and also decrease cost.

Animals ; Caveolin 1 ; genetics ; Deoxyribonucleases ; metabolism ; Gene Amplification ; Humans ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Thionucleotides ; metabolism

Injury or inflammation affecting sensory neurons in the dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spinal central sensitization and neuropathic pain. Recent studies have indicated that, following chronic compression of DRG (CCD) or acute dissociation of DRG (ADD) treatment, both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by activity in cGMP-PKG signaling pathway. Here, we provide evidence supporting the idea that CCD or ADD treatment activates cGMP-PKA signaling pathway in the DRG neurons. The results showed that CCD or ADD results in increase of levels of cGMP concentration and expression of PKG-I mRNA, as well as PKG-I protein in DRG. CCD or ADD treated-DRG neurons become hyperexcitable and exhibit increased responsiveness to the activators of cGMP-PKG pathway, 8-Br-cGMP and Sp-cGMP. Hyperexcitability of the injured neurons is inhibited by cGMP-PKG pathway inhibitors, ODQ and Rp-8-pCPT-cGMPS. In vivo delivery of Rp-8-pCPT-cGMPS into the compressed ganglion within the intervertebral foramen suppresses CCD-induced thermal hyperalgesia. These findings indicate that the in vivo CCD or in vitro ADD treatment can activate the cGMP-PKG signaling pathway, and that continuing activation of cGMP-PKG pathway is required to maintain DRG neuronal hyperexcitability and/or hyperalgesia after these two dissimilar forms of injury-related stress.
Animals ; Cyclic GMP ; analogs & derivatives ; metabolism ; Cyclic GMP-Dependent Protein Kinases ; metabolism ; Ganglia, Spinal ; physiopathology ; Hyperalgesia ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Thionucleotides ; metabolism

BACKGROUNDEndostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually.

METHODSThe artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed.

RESULTSThe cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66 ± 6.79)%, whereas those of the individual ASODN and ES groups were (53.39 ± 3.22)% and (50.22 ± 5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P < 0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study.

CONCLUSIONSOur findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.

Angiogenesis Inhibitors ; administration & dosage ; Animals ; Cell Line, Tumor ; Drug Synergism ; Endostatins ; administration & dosage ; Mice ; Thionucleotides ; administration & dosage ; Urinary Bladder Neoplasms ; pathology

To explore dose-effect or time-effect of vascular endothelial growth factor (VEGF) antisense phosphorothioate oligodeoxynucleotides (AS PS-ODN) on growth of HL-60 cells, and to study the effect mechanism so as to find new role of VEGF, A7, which was the most effective one of AS PS-ODN selected with computer-aided design and experimental assay, contains 20-DNA modified with phosphorothioate and was tranferred into cells mediated with lipofectin. After culture for 72 hours, inhibitive rate of cell growth was detected with MTT methods, viable cells were counted with trypan blue exclusion each 24 hour, cell configuration and apoptosis were observed with Geimsa staining and flow cytometry respectively, level of VEGF protein was detected with VEGF ELISA kit. The results showed that A7 is able to inhibit cell growth of HL-60 in dose-depending manner of AS PS-ODN, to down-regulate VEGF protein expression significantly, and not to induce apoptosis of HL-60 cells. It is concluded that there is possibility that the inhibition effect of VEGF AS PS-ODN on HL-60 cell growth is to restrain cell proliferation without inducing apoptosis of HL-60 cell, which would interpret that endogenous VEGF proteins have a capacity of promoting proliferation of HL-60 cell.
Cell Proliferation ; drug effects ; Flow Cytometry ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Thionucleotides ; pharmacology ; Vascular Endothelial Growth Factor A ; analysis ; physiology

BACKGROUND: Propofol may decrease myocardial contractility via actions on the beta-adrenoceptor-mediated signal transduction. The aim of this study was to evaluate the effect of propofol via beta-adrenoceptor-mediated signal transduction by measuring the tissue levels of cAMP (cyclic adenosine monophosphate). METHODS: The effects of propofol on beta-adrenoceptor mediated cascades were measured with cAMP concentrations, which were stimulated by agonists (l-isoproterenol, GTPgammaS, and forskolin) of each step of beta-adrenoceptor-mediated cascades. RESULTS: While the production of cAMP stimulated by isoproterenol, GTPgammaS, or forskolin are increased (P < 0.05), application of each concentration of propofol (0.1, 1, 10, 100 micrometer) did not alter the levels of cAMP. CONCLUSIONS: Considering that propofol did not alter the tissue cAMP levels when stimulated by isoproterenol, GTPgammaS, and forskolin, propofol appears to have no effect on the beta-adrenoceptor signaling pathway in guinea pig ventricular myocardium.
Adenosine ; Animals ; Forskolin ; GTP-Binding Proteins ; Guanosine 5'-O-(3-Thiotriphosphate) ; Guinea Pigs ; Isoproterenol ; Myocardium ; Propofol ; Signal Transduction

BACKGROUND: Dynorphin A (1-17) is conceived as an endogenous opioid peptide with a high degree of selectivity forkappa- opioid receptor even though it has been reported to sometimes act like amicro- opioid agonist. The aim of this study was to investigate [35S] GTPgammaS binding stimulated activation by dynorphin A (1-17) in the cerebral and thalamic membranes of a rhesus monkey. METHODS: The rhesus monkey (Macaca mulatta, male, n = 1) was euthanized for the preparation of the cerebral and thalamic membranes. Protein concentrations were determined by the Bradford method. In the dynorphin A (1-17)-stimulated [35S] GTPgammaS binding dose-response curve, EC50 (effective concentration 50 nM) and maximum stimulation (% over basal) were determined in the absence or presence of themicro-andkappa-opioid receptor antagonists naloxone (20 nM) and norbinaltorphimine (nor-BNI, 3 nM), respectively. E2078-stimulated [35S] GTPgammaS binding was also determined in the absence or presence ofmicro-andkappa-opioid receptor antagonists in the cortical membrane and compared with dynorphin A (1-17). RESULTS: Values of EC50 and maximum stimulation of dynorphin A (1-17)-stimulated [35S] GTPgammaS binding were as follows: cortex (474 nM/32.0%) and thalamus (423 nM/45.3%). Nor-BNI (3 nM) did not antagonize dynorphin A (1-17)-stimulated [35S] GTPgammaS binding at all in cortical or thalamic membrane, but naloxone (20 nM) produced a 12.2 fold rightward shift of the dynorphin A (1-17)-stimulated [35S] GTPgammaS binding dose-response curve in the thalamic membrane. The EC50 and the maximum stimulation of E2078-stimulated [35S] GTPgammaS binding were 65.6 nM and 22.7%, respectively. In E2078-stimulated [35S] GTPgammaS binding, the dose-response curve was antagonized not by nor-BNI but by naloxone but in the cortical membrane (a 14.2 times rightward shift). CONCLUSIONS: Dynorphin A (1-17) is selective formicro-opioid receptor in agonist-stimulated [35S] GTPgammaS binding in the cortical and thalamic membranes of rhesus monkey.
Dynorphins* ; Guanosine 5'-O-(3-Thiotriphosphate)* ; Haplorhini* ; Humans ; Macaca mulatta ; Male ; Membranes* ; Naloxone ; Opioid Peptides ; Receptors, Opioid ; Thalamus

BACKGROUND: This study was examined whether or not the orphanin FQ (OFQ)-stimulated [35S]GTPgammaS activity interact with DAMGO in the whole brain of mice. METHODS: ICR mice (male, n = 20, 20-25 g) were euthanized for the membrane preparations. In the agonist-stimulated [35S]GTPgammaS binding dose-response curves by OFQ, Ro-64-6198 and DAMGO, the EC50 (effective concentration 50, nM) and maximum stimulation (% over basal) were determined in the presence or absence of J-113397 (10 nM), a NOP (nociceptin-opioid peptide) receptor antagonist. OFQ (1micrometer), Ro-64-6198 (10micrometer), DAMGO (10micrometer) and their combination cocktail were used to determine the interaction between the NOP and MOP (micron-opioid peptide) receptor. RESULTS: The values of EC50 and maximum stimulation of [35S]GTPgammaS binding were as follows: OFQ (9.2 +/- 0.2 nM/17.9 +/- 0.1%), Ro-64-6198 (143.5 +/- 0.5 nM/18.1 +/- 0.4%), and DAMGO (680.6 +/- 0.7 nM/18.1 +/- 0.5%). J-113397 produced a 8.7 and 7.1 fold rightward shifting in the OFQ and Ro-64-6198-stimulated [35S]GTPgammaS binding dose-response curve respectively, but not in the DAMGO. OFQ combined with DAMGO-stimulated [35S]GTPgammaS binding had an additive effect, but not in the OFQ combined with Ro-64-6198. CONCLUSIONS: OFQ, Ro-64-6198 and DAMGO-stimulated [35S]GTPgammaS binding in the brain of mice has receptor selectivity. The [35S]GTPgammaS stimulation of OFQ and DAMGO had an additive effect rather than an anti-opioid effect on the level of intracellular signal transduction through agonist-stimulated [35S]GTPgammaS bindings.
Animals ; Brain* ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Guanosine 5'-O-(3-Thiotriphosphate) ; Membranes ; Mice* ; Mice, Inbred ICR ; Signal Transduction

The effect of antisense oligodeoxyribonucleotides(oligo[dN]s) on hepatitis B virus(HBV) replication in HepG2 cells harboring a cloned HBV genome was examined. Antisense oligo(dN)s directed at translational initiation sites of S, pre C and P genes of HBV were treated to the cells and the amount of HBsAg and HBV DNA content were measured 72 hours after the treatment. HBsAg expressions in HepG2 cells harboring the HBV genome were inhibited 68%, 53%, and 46% by the treatment with antisense oligo[dN] directed at S, pre C, and P gene loci, respectively, and HBV DNA content in the cells was also reduced by the treatment of each antisense oligo[dN]. The doubling times of the cultured cells treated with 25 micrograms, 50 micrograms, and 100 micrograms of antisense oligo[dN]/ml medium were 43.3, 62.1, and 93.0 hours, respectively, compared with 37.5 hours of the untreated control cells. Cellular DNA synthesis was inhibited by the treatment with 100 micrograms/ml of antisense oligo [dN], however, no significant effect was observed by the treatment with 50 micrograms or less of antisense oligo[dN]/ml. These results suggested that antisense oligo[dN]s specific to the translational initiation sites of S, pre C, and P genes of HBV may have therapeutic potential for the suppression of HBV propagation in chronic HBV infected patients.
Cloning, Molecular ; DNA, Viral/*genetics ; Hepatitis B Virus/genetics/*physiology ; Hepatoblastoma/pathology/*virology ; Human ; Liver Neoplasms/pathology/*virology ; Oligonucleotides, Antisense/*pharmacology ; Support, Non-U.S. Gov't ; Thionucleotides/*pharmacology ; Transfection ; Tumor Cells, Cultured/virology ; *Virus Replication

This study was aimed to investigate the possible effects of cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the M(2b) subtype of acute myeloid leukemia (AML-M(2b)) cells. AML-M(2b) is characterized by the non-random chromosome translocation t (8; 21) (q22; q22), through which AML1 (acute myeloid leukemia 1) gene on chromosome 21 is fused with ETO (eight twenty-one) gene on chromosome 8, coding correspondent AML1-ETO fusion protein, which plays a crucial role in the leukemogenesis of AML-M(2b). The AML-M(2b) cell line Kasumi-1 cells were used as an in vitro model. The influences of 8-CPT-cAMP on the proliferation and differentiation of Kasumi-1 cells were evaluated according to cellular morphology, changes in cell surface antigen and cell cycle, as well as nitroblue-tetrazolium (NBT) assay. Meanwhile, semi-quantity RT-PCR and Western blot assay were used to detect the degradation of AML1-ETO fusion protein in Kasumi-1 cells before and after the treatment. The results showed that 8-CPT-cAMP (200 micromol/L) could significantly inhibit cell growth and induce differentiation of Kasumi-1 cells. However, it must be pointed out that 8-CPT-cAMP-induced differentiation in Kasumi-1 is not a typical terminal differentiation. Furthermore, 8-CPT-cAMP exerted little influence on the expression of AML1-ETO fusion gene and its product in Kasumi-1 cells. In conclusion, the 8-CPT-cAMP induced differentiation in Kasumi-1 cells. This results may provide experimental and theoretical basis for the breakthrough of differentiation-induced therapy extended to another leukemia.
Cell Transformation, Neoplastic ; drug effects ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Cyclic AMP ; analogs & derivatives ; pharmacology ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Thionucleotides ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo investigate the effects and related mechanisms of 2-methylthio-adenosine-5'-triphosphate (2-MeSATP), an important extracellular agonist that activates receptors for purine nucleotides (P2XR), on ventricular arrhythmias in rabbits with chronic heart failure (CHF).

METHODSThe male New Zealand rabbits were divide into control (n=12), CHF (n=12) and CHF+2-MeSATP groups (2-MeSATP, n=12). CHF was induced by isoproterenol injection (0.3 mg·kg⁻¹·d⁻¹ for 3 weeks) and rabbits were observed 6 months later. The main cardioelectrophysiological parameters and ventricular arrhythmias were tested by recording monophasic action potential (MAP) with burst-pacing (BCL) in rabbits in vivo. The transient outward potassium current (Ito) was recorded via whole-cell patch clamp technique and the fluorescence intensity of intracellular free Ca²⁺ was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbet ventricular myocytes.

RESULTSCHF rabbits developed severely clinical CHF signs and symptoms, reduced left ventricular ejection fraction and fractional shortening as well as enlarged end-diastolic dimension. Compared with CHF group, APA and MaxdV/dt were significantly increased, while APD20, APD50 and APD90 were significantly reduced in 2-MeSATP group (all P<0.01). Moreover, 2-MeSATP could obviously shorten BCL induced ventricular arrhythmias, and decrease deducibility and persistence time of ventricular arrhythmias with burst-pacing in 2-MeSATP group in vivo (all P<0.05). With voltage clamp model, 2-MeSATP could significantly increase the current density of Ito in different command potential in CHF ventricular myocytes (all P<0.01). When holding potential was set at -50 mV and command potential was set at +50 mV, the current densities of Ito increase was more significant in 2-MeSATP group than that in CHF group ((11.79 ± 4.51) pA/pF vs. (7.94 ± 3.53) pA/pF, P<0.01). 2-MeSATP could completely change the I-V curve upward without changing the I-V curve direction in CHF ventricular myocytes. The fluorescence intensities of intracellular free Ca²⁺ increase was more significant in 2-MeSATP group compared to CHF group ((1 291.98 ± 123.31) µmol/L vs. (793.59 ± 114.65) µmol/L, P<0.01).

CONCLUSION2-MeSATP as a potent agonist acting on P2XR could significantly shorten APD, increase heart rate and improve cardiac performance as well as decrease the susceptibility of ventricular arrhythmias in this rabbit CHF model. Our results suggest that Ito increase and sarcoplasmic reticulum uptake Ca²⁺ enhancement as well as dynamic balance of intracellular Ca²⁺ cycling sustenance might linked to the beneficial effects of 2-MeSATP in this CHF model.

Action Potentials ; Adenosine Triphosphate ; analogs & derivatives ; Animals ; Brugada Syndrome ; Cardiac Conduction System Disease ; Chronic Disease ; Heart Failure ; Heart Ventricles ; Isoproterenol ; Male ; Myocytes, Cardiac ; Patch-Clamp Techniques ; Potassium ; Rabbits ; Thionucleotides