Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Scutellarin attenuates endothelium-dependent aasodilation impairment induced by hypoxia reoxygenation, through regulating the PKG signaling pathway in rat coronary artery / 中国天然药物

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.

2-Methylthio-adenosine-5'-triphosphate inhibits ventricular arrhythmogenesis in rabbits with chronic heart failure / 中华心血管病杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>CHF 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).</p><p><b>CONCLUSION</b>2-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.</p>

Enhancement of GABA-activated currents by arginine vasopressin in rat dorsal root ganglion neurons / 生理学报

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.

Monitoring Thiopurine Metabolites in Korean Pediatric Patients with Inflammatory Bowel Disease

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.

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endostatin 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>Our 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.</p>

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 / 生理学报

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.

Pharmacological characteristics of contractile responses regulated by P2Y receptors in circular smooth muscle of the rat gastric body / 药学学报

This study is to observe the difference in pharmacological characteristics between circular smooth muscles of rat isolated gastric body and gastric fundus, and to investigate the effects of nucleoside and nucleotide on circular smooth muscle of the rat gastric body and the involved receptors. Circular muscle strips of the rat gastric body and gastric fundus were prepared, and contractile responses to agonists were investigated with a technique of drug-receptor interaction in functional system. There was no significant difference between the circular muscle strips of the gastric body and gastric fundus in the responses to KCl, and no difference in EC50 values of contractile responses for 5-HT and His between the two kinds of preparations (P > 0.05). However, Emax values of contractile responses to 5-HT and His [(0.81 +/- 0.26) and (0.88 +/- 0.27) g] in gastric body were significantly smaller than those in gastric fundus [(2.67 +/- 0.61) and (1.90 +/- 0.68) g, P < 0.01], and EC50 value of CCh produced contractile response [(0.45 +/- 0.15) micromol x L(-1)] in gastric body was significantly higher than that in gastric fundus [(0.20 +/- 0.09) micromol x L(-1), P < 0.01]. In precontracted circular muscle strips of the gastric body, ATP (0.1-3000 micromol x L(-1)) produced only a contractile response concentration-dependently, but the same concentration of ATP induced a biphasic response (relaxation followed by a contraction) in precontracted circular muscle strips of the gastric fundus. ATP, UTP, ADP, 2-MeSATP and alpha,beta-MeATP produced contractile responses concentration-dependently in circular muscle strips of the rat gastric body. The EC50 value for 2-MeSATP [(7.2 +/- 5.2) nmol x L(-1)] was about 500 times lower than that for Ach [(3.47 +/- 1.20) micromol x L(-1)]. The rank order of potency for the contraction was 2-MeSATP>ADP>ATP=UTP>alpha,beta-MeATP>adenosine. The contractile responses to ATP and UTP were not significantly affected by phentolamine, propranolol, atropine or tetrodotoxin. In conclusion, there is a significant difference in pharmacological characteristics between the circular smooth muscles of the rat gastric body and gastric fundus and nucleotides might be important mediators responsible for the contraction via a specific P2Y receptor in circular smooth muscle of the rat gastric body.

cAMP analogue 8-CPT-cAMP inducing differentiation in the M2b subtype of acute myeloid leukemia cell line Kasumi-1 / 中国实验血液学杂志

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.

Involvement of Thromboxane A2 in the Modulation of Pacemaker Activity of Interstitial Cells of Cajal of Mouse Intestine

Although many studies show that thromboxane A2 (TXA2) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA2 on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA2 in ICC with whole cell patch-clamp technique. Externally applied TXA2 (5 micrometer) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA2 were inhibited by intracellular application of GDP-beta-S. The pretreatment of ICC with Ca2+ free solution and thapsigargin, a Ca2+-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA2-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA2-induced effects on pacemaker currents. These results suggest that TXA2 can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA2 may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca2+ modulation.

Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 micrometer) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-beta S that showed maximal IL-10 release were 100, 10, 100, and 100 micrometer respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP > 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down- regulated by either MRS2179 (a P2Y1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y1 and P2Y11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 micrometer) was restored by TNP-ATP (an antagonist of the P2X1, P2X3, and P2X4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y12 receptor antagonist) or pertusis toxin (PTX; a Gi protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y11 receptor or the P2Y1 receptor, respectively.

Inhibition of telomerase with human telomerase reverse transcriptase antisense enhances tumor necrosis factor-alpha-induced apoptosis in bladder cancer cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis.</p><p><b>METHODS</b>Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.</p><p><b>RESULTS</b>AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-alpha while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.</p><p><b>CONCLUSIONS</b>AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.</p>

Preparation of oligodeoxynucleotide encapsulated cationic liposomes and release study with models of cellular membranes

Cationic liposomes are used for cellular delivery of antisense oligodeoxynucleotide [AsODN], where release of encapsulated AsODN is mainly controlled by endocytosis and fusion mechanisms. In this investigation, it was tried to model such a release process that is difficult to evaluate in cell culture. For this purpose, an AsODN model [against protein kinase C-alpha] was encapsulated in a DODAP-containing cationic liposome and evaluated for size, zeta-potential, encapsulation and ODN stability. Vesicular models of outer layer and total plasma membranes and early and late endosomal membranes were developed, based on lipid content and pH, using ether injection method. ODN release was determined by the fluorescence dequenching of encapsulated FITC-ODN. Zeta potential, size and ODN encapsulation efficiency of the prepared liposomes were -2.49 +/- 7.15 mV, 108.4 nm and 73% respectively. ODN protection was 3-4 times more than that of conventional liposome/ODN complexation method. There was a correlation between model concentration and percent of ODN release. At 7.5 Mu M, the percent of released ODN was 76% for the cholesterol-free model of the late endosome and 16% for the early endosomal membrane; while the release was less than 11% for the models of plasma membrane. ODN release increased with temperature in the range of 4-37°C for the late endosomal model, but not for others, possibly due to their high cholesterol contents or acidic pH. The interaction was fast and completed within 5 minutes and didn't change in the range of 5-60 minutes. Our data are in agreement with published cell culture studies and reveal that cell-liposomes interaction can be modeled by lamellar membranes

A reverse transcriptase-polymerase chain reaction method improving the specific amplification of caveolin-1 gene sequences / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct a reverse transcriptase-polymerase chain reaction (RT-PCR)approach that can improve the specificity of primers while dropping down the nonspecific amplification.</p><p><b>METHODS</b>In 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.</p><p><b>RESULTS</b>Taq 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.</p><p><b>CONCLUSION</b>Compared 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.</p>

Effect of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptosis and thermosensitivity of BcaCD885 cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effect of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptosis and thermosensitivity of BcaCD885 cells.</p><p><b>METHODS</b>After 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Phosphorothioate antisense Bcl-xL oligodeoxynucleotides can induce apoptosis and improve thermosensitivity of BcaCD885 cells.</p>

Effect and mechanism of VEGF antisense phosphorothioate deoxynucleotides on HL-60 leukemic cells / 中国实验血液学杂志

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.

Effects of purinergic analogues on spontaneous contraction and electrical activities of gastric antral circular muscle in guinea-pig / 生理学报

In order to investigate the role of non-adrenergic non-cholinergic nerves in regulating mechanical and electrical activity of gastric circular smooth muscle, the effects of ATP and its analogues on gastric motility and electrical activities were observed in guinea-pig. In organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured. Electrical activity of the muscle was recorded by using intracellular microelectrode. Electrical and mechanical activities were recorded by chart recorder. ATP and 2-MeSATP potentiated the mechanical activity but did not affect electrical activity in gastric circular smooth muscle. ATP and 2-MeSATP-induced contraction was effectively blocked by nonselective P2y-purinoceptor antagonist, reactive-blue-2 and suramin, but ATP-induced contraction was not blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. However, alpha,beta-MeATP, P2x-purinoceptor agonist, attenuated slow waves with membrane hyperpolarization and inhibited contraction. The relaxation by beta,gamma-MeATP was blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. ATP-induced contraction was blocked by external calcium-free, but not by nicardipine, a L-type calcium channel blocker. Indomethacin, a nonselective cyclooxygenase inhibitor, did not block ATP-induced contraction. The results suggest that: (1) ATP- and analogues-induced contraction is mediated by P2y-purinoceptor, whereas alpha,beta-MeATP-induced relaxation by P2x-purinoceptor in guinea-pig gastric antral circular smooth muscle. (2) ATP-induced contraction is dependent on extracellular calcium, but Ca2+ entry is not mediated by L-type calcium channel. (3) Prostaglandins are not involved in ATP- and analogue-induced contraction of gastric circular smooth muscle in guinea-pigs, and alpha,beta-MeATP-induced relaxation is related to membrane hyperpolarization.

Oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation / 药学学报

<p><b>AIM</b>To explore whether the oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation.</p><p><b>METHODS</b>Intracellular mean fluorescence intensity was measured by flow cytometry.</p><p><b>RESULTS</b>After 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.</p><p><b>CONCLUSION</b>Hematological 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.</p>

In vitro study of the effects of arsenic trioxide combined with 8-CPT-cAMP on differentiation induction in retinoic acid resistant acute promyelocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the potential effects of arsenic trioxide (As(2)O(3)) combined with 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.</p><p><b>METHODS</b>The RA resistant APL cell lines NB4-R1 and NB4-R2 were used as in vitro models. The effect of As(2)O(3) and/or 8-CPT-cAMP was evaluated according to cellular morphology, cell surface antigen and nitroblue-tetrazolium (NBT) assay. Meanwhile, immunofluorescence analysis and Western blot assay were used to detect the degradation of PML-RAR alpha fusion protein and the change of several key cell cycle regulatory proteins in these cells before and after the treatment.</p><p><b>RESULTS</b>Low dose of As(2)O(3) (0.25 micromol/L) synergized with 8-CPT-cAMP (200 micromol/L) in inducing differentiation of NB4-R1 and NB4-R2 cells, while neither of these two drugs alone could induce differentiation of these cells. In addition, 8-CPT-cAMP was able to inhibit the cell growth by modulating the expression of some important cell cycle regulators and to facilitate the As(2)O(3)-mediated degradation of PML-RAR alpha fusion protein.</p><p><b>CONCLUSIONS</b>As(2)O(3) combined with 8-CPT-cAMP could induce differentiation of RA-resistant APL cells.</p>

Effect of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.</p><p><b>METHODS</b>Using 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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSIONS</b>Dual 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.</p>