1.A reverse transcriptase-polymerase chain reaction method improving the specific amplification of caveolin-1 gene sequences.
Yang-yan XU ; Hui-ling YANG ; Yong YOU ; Li QIN ; Jian TU
Chinese Journal of Medical Genetics 2006;23(1):59-62
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
2.Activation of cGMP-PKG signaling pathway contributes to neuronal hyperexcitability and hyperalgesia after in vivo prolonged compression or in vitro acute dissociation of dorsal root ganglion in rats.
Zhi-Jiang HUANG ; Hao-Chuan LI ; Su LIU ; Xue-Jun SONG
Acta Physiologica Sinica 2012;64(5):563-576
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
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Cyclic GMP
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analogs & derivatives
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metabolism
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Cyclic GMP-Dependent Protein Kinases
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metabolism
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Ganglia, Spinal
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physiopathology
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Hyperalgesia
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physiopathology
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Rats
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Rats, Sprague-Dawley
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Signal Transduction
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Thionucleotides
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metabolism
3.Monitoring Thiopurine Metabolites in Korean Pediatric Patients with Inflammatory Bowel Disease.
Mi Jin KIM ; Soo Youn LEE ; Yon Ho CHOE
Yonsei Medical Journal 2014;55(5):1289-1296
PURPOSE: This study aimed to assess the role of thiopurine S-methyltransferase (TPMT) and 6-thioguanine nucleotide (6-TGN) as predictors of clinical response and side effects to azathioprine (AZA), and estimate the optimal AZA dose in Korean pediatric inflammatory bowel disease (IBD) patients. MATERIALS AND METHODS: One hundred and nine pediatric IBD patients in whom AZA treatment was required were enrolled. Thiopurine metabolites were monitored since September 2010. Among them, 83 patients who had prescribed AZA for at least 3 months prior to September 2010 were enrolled and followed until October 2011 to evaluate optimal AZA dose, adverse effects and disease activity before and after thiopurine metabolite monitoring. RESULTS: The result of the TPMT genotype was that 102 patients were *1/*1 (wild type), four were *1/*3C, one was *1/*6, one was *1/*16 (heterozygote) and one was *3C/*3C (homozygote). Adverse effects happened in 31 patients pre-metabolite monitoring and in only nine patients post-metabolite monitoring. AZA dose was 1.4+/-0.31 mg/kg/day before monitoring and 1.1+/-0.46 mg/kg/day after monitoring (p<0.001). However, there were no statistical differences in disease activity during metabolite monitoring period (p=0.34). Adverse effects noticeably decreased although reduction of the AZA dose since monitoring. CONCLUSION: TPMT genotype and thiopurine metabolite monitoring could be helpful to examine TPMT genotypes before administering AZA and to measure 6-TGN concentrations during prescribing AZA in IBD patients.
Adolescent
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Adult
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Azathioprine/*adverse effects/therapeutic use
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Child
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Child, Preschool
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Female
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Genotype
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Guanine Nucleotides/metabolism
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Humans
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Inflammatory Bowel Diseases/*drug therapy/metabolism
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Male
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Methyltransferases/genetics/metabolism
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Republic of Korea
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Risk Factors
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Thionucleotides/metabolism
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Treatment Outcome
4.cAMP analogue 8-CPT-cAMP inducing differentiation in the M2b subtype of acute myeloid leukemia cell line Kasumi-1.
Qi ZHU ; Jun-Pei HU ; Pei-Min JIA ; Zhen-Yi WANG ; Jian-Hua TONG
Journal of Experimental Hematology 2008;16(1):44-47
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
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drug effects
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Core Binding Factor Alpha 2 Subunit
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genetics
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metabolism
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Cyclic AMP
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analogs & derivatives
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pharmacology
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Humans
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Leukemia, Myeloid, Acute
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genetics
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metabolism
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pathology
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Oncogene Proteins, Fusion
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genetics
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metabolism
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RUNX1 Translocation Partner 1 Protein
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Thionucleotides
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pharmacology
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Tumor Cells, Cultured
5.Enhancement of GABA-activated currents by arginine vasopressin in rat dorsal root ganglion neurons.
Fang QIU ; Wang-Ping HU ; Zhi-Fan YANG
Acta Physiologica Sinica 2014;66(6):647-657
A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹⁰-1 × 10⁻⁵ mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻⁶ mol/L). Also it could be removed by intracellular dialysis of either GDP-β-S (5 × 10⁻⁴mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻⁶ mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.
Animals
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Arginine Vasopressin
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pharmacology
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Ganglia, Spinal
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cytology
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Guanosine Diphosphate
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analogs & derivatives
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pharmacology
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Indoles
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Maleimides
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Membrane Potentials
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Neurons
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drug effects
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Patch-Clamp Techniques
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Rats
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Rats, Sprague-Dawley
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Receptors, GABA-A
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metabolism
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Signal Transduction
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Thionucleotides
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pharmacology
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gamma-Aminobutyric Acid
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pharmacology
6.The effect of telomerase inhibitors on oral squamous carcinoma cells.
Zhiqiang YANG ; Wen CHEN ; Qiao ZHANG
West China Journal of Stomatology 2003;21(3):231-234
OBJECTIVETo study the potential anti-tumor effect of telomerase inhibitors.
METHODSHuman oral squamous carcinoma cell line KB was selected as target cell. The effects of 3'-azido-3'-deoxythymidine (AZT) and human antisense phosphorothioate (AS-ONS) for telomerase template on KB cell line were investigated. The cytotoxic effect of AZT and AS-ONS on tumor cells was quantified using MTT colorimetric assay. Assay of 3H-TdR incorporation was undertaken to measure the cell proliferation. The changes of telomerase activity after treatment was detected and quantified by telomeric repeat amplification protocol (TRAP) semi-quantitative analysis. The Flow Cytometry was used to detect apoptosis and measure cell cycle.
RESULTSBoth AZT and AS-ONS inhibited the growth of KB cell line in a certain range of concentrations, and meanwhile the telomerase activity was reduced after treatment. In addition, both AZT and AS-ONS can induce apoptosis and arrest G1 phase of cell cycle.
CONCLUSIONThe results obtained above indicated that AZT and AS-ONS could be potentially used as an anti-oral carcinoma agent or an auxiliary treatment for cancer. Those inhibitory effects might be partially due to the induction of apoptosis and the prolongation of cell cycle.
Antineoplastic Agents ; pharmacology ; Carcinoma, Squamous Cell ; pathology ; Cell Division ; drug effects ; Enzyme Inhibitors ; pharmacology ; Humans ; Mouth Neoplasms ; pathology ; Oligodeoxyribonucleotides ; pharmacology ; Telomerase ; antagonists & inhibitors ; metabolism ; Thionucleotides ; Tumor Cells, Cultured ; Zidovudine ; pharmacology
7.Oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation.
Yuan-gui ZHU ; Guang-sheng ZHUO ; Zhi-zhe CHEN ; Xiao-chun CHEN
Acta Pharmaceutica Sinica 2003;38(6):401-404
AIMTo explore whether the oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation.
METHODSIntracellular mean fluorescence intensity was measured by flow cytometry.
RESULTSAfter treatment with FITC-labeled G3139 at the concentration of 0.60 mumol.L-1 for 4 h, the G3139 uptake into peripheral blood mononuclear cell and bone marrow mononuclear cell in hematological tumor patients was significantly higher than that in normal control. There was different uptake of G3139 among the malignant hematological tumor cell strains, and the uptake in cells derived from monocyte, B lymphocyte and myeloid cell was much higher than that in cells derived from T lymphocyte. After treatment with all-trans retinoic acid (ATRA), HL60 cell proliferation was markedly inhibited and the uptake of G3139 decreased significantly.
CONCLUSIONHematological tumor cells were capable of taking up oligonucleotide, and the oligonucleotide uptake in hematological tumor cells is related to its cellular species and its activation.
Biological Transport ; Cell Division ; physiology ; Genes, bcl-2 ; genetics ; HL-60 Cells ; Humans ; Leukemia ; metabolism ; pathology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Leukocytes, Mononuclear ; metabolism ; Lymphoma, Non-Hodgkin ; metabolism ; pathology ; Oligonucleotides, Antisense ; metabolism ; Thionucleotides ; metabolism ; Tretinoin ; pharmacology ; Tumor Cells, Cultured
8.Effect of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.
Cai-ying YE ; Qing-xia LI ; Xiao-li YU ; Jing-jing QI ; Juan LI ; De-chang ZHANG
Acta Academiae Medicinae Sinicae 2003;25(3):297-300
OBJECTIVETo observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.
METHODSUsing whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution.
RESULTS(1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists.
CONCLUSIONSDual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.
Animals ; Enkephalin, D-Penicillamine (2,5)- ; pharmacology ; GTP-Binding Proteins ; physiology ; Glioma ; metabolism ; pathology ; Guanosine Monophosphate ; analogs & derivatives ; pharmacokinetics ; Hybrid Cells ; metabolism ; pathology ; Mice ; Neuroblastoma ; metabolism ; pathology ; Patch-Clamp Techniques ; Pertussis Toxin ; pharmacology ; Potassium Channels, Inwardly Rectifying ; metabolism ; Rats ; Receptors, Opioid ; agonists ; Thionucleotides ; pharmacokinetics
9.Scutellarin attenuates endothelium-dependent aasodilation impairment induced by hypoxia reoxygenation, through regulating the PKG signaling pathway in rat coronary artery.
Ya-Juan CHEN ; Lei WANG ; Guang-Yu ZHOU ; Xian-Lun YU ; Yong-Hui ZHANG ; Na HU ; Qing-Qing LI ; Chen CHEN ; Chen QING ; Ying-Ting LIU ; Wei-Min YANG
Chinese Journal of Natural Medicines (English Ed.) 2015;13(4):264-273
Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Animals
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Apigenin
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pharmacology
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Cell Adhesion Molecules
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drug effects
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Cell Hypoxia
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Coronary Vessels
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drug effects
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Cyclic GMP
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analogs & derivatives
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metabolism
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pharmacology
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Cyclic GMP-Dependent Protein Kinases
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Glucuronates
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pharmacology
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Microfilament Proteins
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drug effects
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NG-Nitroarginine Methyl Ester
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metabolism
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pharmacology
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Phosphoproteins
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drug effects
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Rats
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Rats, Sprague-Dawley
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Reperfusion Injury
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complications
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physiopathology
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Signal Transduction
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drug effects
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Thionucleotides
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metabolism
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pharmacology
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Vasodilation
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drug effects
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physiology
10.Effect of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptosis and thermosensitivity of BcaCD885 cells.
Yong-wen HE ; Li BIAN ; Xin-hua LIANG
West China Journal of Stomatology 2004;22(2):112-114
OBJECTIVETo study the effect of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptosis and thermosensitivity of BcaCD885 cells.
METHODSAfter phosphorothioate antisense Bcl-xL oligodeoxynucleotides were transfected into BcaCD885 cells. The characteristics of apoptotic cells were evaluated by morphological observation and TUNEL staining. Apoptotic rate and Bcl-xL protein expression were analyzed with flow cytometry. The influence of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptotic rate of BcaCD885 cells induced by hyperthermia with 43 degrees C 40 min was also examined through flow cytometry.
RESULTSThe BcaCD885 cells transfected with phosphorothioate antisense Bcl-xL oligodeoxynucleotides displayed apoptotic morphological features. The Bcl-xL protein expression level of these cells was down-regulated significantly compared with the controlled group (P < 0.05). The apoptotic rate of these cells induced by hyperthermia was increased significantly compared with the controlled group (P < 0.05).
CONCLUSIONPhosphorothioate antisense Bcl-xL oligodeoxynucleotides can induce apoptosis and improve thermosensitivity of BcaCD885 cells.
Apoptosis ; drug effects ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Hot Temperature ; Humans ; Hyperthermia, Induced ; Mouth Neoplasms ; genetics ; pathology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Thionucleotides ; pharmacology ; Transfection ; Tumor Cells, Cultured ; bcl-X Protein