摘要:
The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.
摘要:
Lipoprotein lipase (LPL) is a rate-limiting enzyme that hydrolyzes circulating triglyceride-rich lipoproteins such as very low-density lipoproteins and chylomicrons. A decrease in LPL activity is associated with an increase in plasma triglycerides (TG) and a decrease in plasma high-density lipoprotein cholesterol (HDL-C). The increase in plasma TG and decrease in plasma HDL-C are risk factors for cardiovascular disease. Tsutsumi et al. hypothesized that elevating LPL activity would cause a reduction of plasma TG and an increase in plasma HDL-C, resulting in protection against the development of atherosclerosis. To test this hypothesis, Otsuka Pharmaceutical Factory, Inc. synthesized the LPL activator NO-1886. NO-1886 increased LPL mRNA and LPL activity in adipose tissue, myocardium and skeletal muscle, resulting in an elevation of postheparin plasma LPL activity and LPL mass in rats. NO-1886 also decreased plasma TG concentration and caused a concomitant rise in plasma HDL-C. Long-term administration of NO-1886 to rats and rabbits with experimental atherosclerosis inhibited the development of atherosclerotic lesions in coronary arteries and aortas. Multiple regression analysis suggested that the increase in plasma HDL-C and the decrease in plasma TG protect from atherosclerosis. The atherogenic lipid profile is changed to an antiatherogenic profile by increasing LPL activity, resulting in protection from of atherosclerosis. Therefore, the LPL activator NO-1886 or other possible LPL activating agents are potentially beneficial for the treatment of hypertriglyceridemia, hypo-HDL cholesterolemia, and protection from atherosclerosis.
期刊:
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE,2003年11(6):767-771 ISSN:1107-3756
通讯作者:
Liu, ZC
作者机构:
Sun Yat Sen Univ, Inst Canc, Ctr Canc, Guangzhou 510060, Peoples R China.;Nanhua Univ, Inst Pharmacol, Hengyang 421001, Peoples R China.;[Liu, ZC] Sun Yat Sen Univ, Inst Canc, Ctr Canc, 651 DongFeng Rd E, Guangzhou 510060, Peoples R China.
通讯机构:
[Liu, ZC] S;Sun Yat Sen Univ, Inst Canc, Ctr Canc, 651 DongFeng Rd E, Guangzhou 510060, Peoples R China.
摘要:
Manumycin was reported to have inhibitory effect on farnesyltransferase by competing with the farnesyl pyrophosphate substrate. It exhibited different antiproliferative activity in human hepatocellular carcinoma HepG2 cells, primary cultured human cardiac muscle cells and human liver cells (CLC). HepG2 cells overexpressing ras gene were more sensitive to manumycin than the other cells. The difference might be related to Ras protein levels in these cell lines. Manumycin reduced the amount of functional ras localized at the cytoplasmic membrane, resulting in blocked C-raf-1 assocation with Ras. Manumycin inhibited ERK1/2 phosphorylation in HepG2 cells without reduced expression of ERK1/2 protein. The levels of protein MKP-1 were significantly up-regulated. Our study also demonstrated that manumycin inhibited p85/PI3K and Akt phosphorylation without reduced expression of p85/PI3K and Akt, and interfered with the association of p85/PI3K and Ras. These findings indicated that manumycin interfered with Ras membrane localization, shut down the downstream pathways of Ras and inhibited cell proliferation in HepG2 cells.
期刊:
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE,2003年12(6):955-959 ISSN:1107-3756
通讯作者:
Zhou, JM
作者机构:
Sun Yat Sen Univ, Inst Canc, Ctr Canc, Guangzhou 510060, Peoples R China.;Nanhua Univ, Inst Pharmacol, Hengyang 421001, Peoples R China.;[Zhou, JM] Sun Yat Sen Univ, Inst Canc, Ctr Canc, 651 DongFeng Rd E, Guangzhou 510060, Peoples R China.
通讯机构:
[Zhou, JM] S;Sun Yat Sen Univ, Inst Canc, Ctr Canc, 651 DongFeng Rd E, Guangzhou 510060, Peoples R China.
关键词:
hepatocellular carcinoma;manumycin;apoptosis
摘要:
Farnesyltransferase inhibitors (FTIs) were developed to prevent Ras processing and thus to be effective agents for the treatment of cancers harbouring mutated ras. In the present study, HepG2 cells underwent internucleosomal DNA fragmentation after treatment with farnesyltransferase inhibitor manumycin (20 muM) for 12 h. Flow cytometric analysis showed that HepG2 cells were accumulated in the G(2)/M phase of the cell cycle and the number of apoptotic sub-G1 fraction of cells was increased after treatment with manumycin in a time-dependent manner. During the induction of apoptosis, expression of p53 and p21(WAF1) was upregulated, phosphorylation of IkappaB-alpha was blocked, caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B were cleaved, and Bcl-2 and Bax protein expression remained unchanged. These results indicated that manumycin induced apoptosis in HepG2 cells. The induction of apoptosis by manumycin involved the upregulation of p53 and p21(WAF1), the activation of caspases, and the inhibition of nuclear factor-kappaB (NF-kappaB) pathway. However, Bcl-2 and Bax are not associated with manumycin-mediated apoptosis.
作者机构:
Nanhua Univ, Inst Pharmacol & Pharm, Inst Biotechnol, Hengyang 421001, Peoples R China.;Charles R Drew Univ Med & Sci, Vasc Biol Lab, Los Angeles, CA 90059 USA.;Jinan Univ, Inst Life Sci & Biotechnol, Guangzhou, Peoples R China.;Genomapping Inc, Tianjin, Peoples R China.;[Zhang, J; Liao, DF; Fang, WY] Institute of Biotechnology, Institute of Pharmacology and Pharmacy, Nanhua University, Hengyang, China
通讯机构:
[Kai Li] I;Institute of Biotechnology, Institute of Pharmacology and Pharmacy, Nanhua University, Hengyang, China<&wdkj&>Vascular Biology Laboratory, Charles R. Drew University of Medicine and Science, Los Angeles
关键词:
mutagenesis;single nucleotide polymorphisms
摘要:
DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3' ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.
摘要:
AIM: To identify the specific serine/threonine residues in the C-terminal tail of thromboxane receptor α (TPα)being phosphorylated and desensitized, and various alanine mutants of these serine/threonine residues were checkedfor their ability to serve as substrates. METHODS: To facilitate the identification of the intracellular domainsinvolved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used assubstrates for the purified PKC, and then the cDNA of phosphorylated protein was mutagenized to localize themajor site of receptor phosphorylation induced by protein kinase C. Human embryonic kidney (HEK) 293 cellsstably transfected with the His-tagged wild type or mutant TPα were used to study the phosphorylation anddesensitization. RESULTS: Only the C-terminal tail can be used as a substrate for the purified PKC. Set-331(mP4) was demonstrated to be heavily phosphorylated, Ser-324 (mP1) was shown to be slightly phosphorylated,Ser-329 was illustrated to be faintly phosphorylated, and other Ser/Thr residues were not found to be phosphorylated.Phorbol-12-myristate-13-acetate (PMA) induced receptor phosphorylation in HEK 293 cells expressing the wildtype TPα. However, PMA did not significantly trigger receptor phosphorylation in I-IEK 293 cells expressing theS331A mutant receptor. Pretreatment of the cells expressing the wild type with PMA inhibited I-BOP induced Ca~(2+) release, however, pretreatment of the cells expressing the S33 lA mutant receptor with PMA did not abolish I-BOP induced Ca~(2+) release. CONCLUSION: Ser-331 is the major and crucial site of receptor phosphorylation and desensitization.
摘要:
The synthetic compound NO-1886 ([4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]-phosphonic acid diethyl ester, CAS 133208-93-2) is a lipoprotein lipase activator which decreases plasma triglycerides and elevates high-density lipoprotein cholesterol (HDL-C) levels. However, the effects of NO-1886 on plasma glucose level and atherosclerosis in diabetes are not clear. The aim of this study was to ascertain whether the compound lowers plasma glucose and suppresses atherosclerosis in New Zealand White rabbits with high fat/high sucrose-induced mild diabetes. High fat/high sucrose feeding increased plasma total cholesterol, triglyceride and glucose levels and decreased HDL-C levels resulting in atherosclerosis in the aorta. Administration of NO-1886 to the rabbits resulted in decreased plasma total cholesterol, triglyceride and glucose levels and increased HDL-C levels after 20 weeks of treatment. Furthermore, NO-1886 provided protection against the development of atherosclerosis in the aorta. These data indicate that NO-1886 not only ameliorates the lipid disorder, but also lowers plasma glucose levels and suppresses atherosclerosis in the aorta of diabetic rabbits.
摘要:
AIM: To investigate the mechanism by which probucol (PBC) affected adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). METHODS: Effects of PBC on expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, a nd E-selectin in human umbilical vein endothelial cells were examined. Moreover, the inhibitory effect of PBC were compared with that of monoclonal antibodies (mAbs) to ICAM-1, VCAM-1, P-selectin, and E-select in on adhesion induced by oxidized-low density lipoprotein(Ox-LDL). RESULTS: PBC at 10 to 80 micromol/L inhibited Ox-LD L-induced adhesion index from 16.7 % to 7.0 % (P < 0.01) and Ox-LDL-induced expression of ICAM-1 (75 %) and P-selectin (72 %). mAbs to ICAM -1 or P-selectin, when used alone, could only slightly reduce the adhesion of monocyte to HUVEC. When both monoclonal antibodies were used in combination, the adhesion was markedly inhibited from 16.7 % to 11.3 % (P < 0.01), but the effect was still weaker than that of PBC (average 9.3 %). CONCLUSION: PBC exerts its inhibitory effect on the adhesion of monocyte to HUVEC by inhibiting the expression of ICAM-1 and P-selectin.
摘要:
Objective. To study the features of vascular smooth muscle cell (VSMC) proliferation induced by endothelin-1 (ET-1). Methods. VSMCs of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats were cultured and treated with ET-1. Basic fibroblast growth factor (bFGF) gene expression was measured using both Northern blot and an enzyme-linked immunoassay. Results. ET-1 resulted in an increase in bFGF transcripts at 8-24 h; bFGF levels were significantly higher in VSMCs treated with ET-1 than in those not treated. However, VSMCs growth responses in SHR and WKY were different. Smooth muscle cells of SHR were hyper-responsive to ET-1. Maximal bFGF mRNA levels were elevated 3.5-fold at 4 h of stimulation in WKY and 8-fold at 8h in SHR4. Moreover, the proliferation of VSMCs induced by ET-1 was inhibited by antisense phosphorothioate oligodeoxynucleotides (10 μmol/L AS-bFGF) but not sense bFGF oligomers at the same concentrations, being reduced by 80% in SHR and 40% in WKY vs control, respectively. Furthermore, the effect of AS-bFGF oligomers on SHR SMC proliferation is significantly greater than on WKY SMC proliferation. Conclusion. ET-1 may be required for exaggerated vascular growth responses in SHR and bFGF may be involved.
摘要:
Objective To study the resistance mechanism of clinical isolates of Ureaplasma urealyticum resistant to fluoroquinolones. Methods Thirteen isolates of Ureaplasma urealyticum resistant to six fluoroquinolones were selected out of 184 clinical isolates and their QRDRs (quinolone resistance-determining region) gyrA, gyrB, parC and parE were amplified by PCR. Sequencing results were compared to those susceptible reference strains and a comparison of deduced amino acid sequences were performed. Results Sequence comparison revealed a C to A change at 87nt of gyrA QRDR leading to the substitution of Asp95 with glutamic acid and a C to T change at 50nt of parC QRDR leading to the substitution of Ser8O with leucine. Conclusion These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with fluoroquinolone resistance of Ureaplasma urealyticum.
摘要:
AIM: To investigate the mechanism by which probucol (PBC) affected adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). METHODS: Effects of PBC on expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and E-selectin in human umbilical vein endothelial cells were examined. Moreover, the inhibitory effect of PBC were compared with that of monoclonal antibodies (mAbs) to ICAM-1, VCAM-1, P-selectin, and E-selectin on adhesion induced by oxidized-low density lipoprotein (Ox-LDL). RESULTS: PBC at 10 to 80 μmol/L inhibited Ox-LDL induced adhesion index from 16.7 % to 7.0 % (P < 0.01) and Ox-LDL-induced expression of ICAM-1 (75 %) and P-selectin (72 %). mAbs to ICAM-1 or P-selectin, when used alone, could only slightly reduce the adhesion of monocyte to HUVEC. When both monoclonal antibodies were used in combination, the adhesion was markedly inhibited from 16.7 % to 11.3 % (P < 0.01), but the effect was still weaker than that of PBC (average 9.3 %). CONCLUSION: PBC exerts its inhibitory effect on the adhesion of monocyte to HUVEC by inhibiting the expression of ICAM-1 and P-selectin.
作者机构:
[Wan, LX] Nanhua Univ, Res Ctr Mol Biol, Hengyang 421001, Peoples R China.;Univ Hong Kong, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China.;Sch Med, Dept Pharmacol, New Haven, CT 06510 USA.;Cent S Univ, Dept Pharmacol, Changsha 410086, Peoples R China.
通讯机构:
[Wan, LX] N;Nanhua Univ, Res Ctr Mol Biol, Hengyang 421001, Peoples R China.
关键词:
human scavenger receptor A;transgenic mice;endothelial cells;atherosclerosis
摘要:
OBJECTIVES: To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo. METHODS: Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-AI expression in transgenic mice. The activity of human SR-AI was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy. RESULTS: The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma I digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl II digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and immunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmission electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells. CONCLUSIONS: A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transgenic model for investigation of atherosclerosis and functions of human SR-A.
作者机构:
[Chen, WZ] Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai 200031, Peoples R China.;Hengyang Med Coll, Dept Pharmacol, Hengyang 421001, Peoples R China.
通讯机构:
[Chen, WZ] C;Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai 200031, Peoples R China.
关键词:
Eposprostenol;Flavones;Ginggolides;Ginkgo biloba;Lysophosphatidylcholines;Malondialdehyde;Thoracic aorta;Vascular endothelium;Vitamin E
摘要:
AIM: To study the protective effects of Ginkgo biloba extract (GbE) against endothelial cell damage induced by lysophosphatidylcholine (LPC). METHODS: The vasorelaxation response to acetylcholine (ACh) were investigated in the isolated rabbit thoracic aorta. Lipid peroxidation products were determined by measuring thiobarbituric acid reactive substance. RESULTS: GbE attenuated the inhibition of vasorelaxation response to ACh and prevented the LPC-induced increase of malondialdehyde (MDA) content both in thoracic aortae. GbE prevented the leakage of LDH and the increase of MDA content in cultured endothelial cells in a concentration-dependent manner. GbE also markedly increased epoprostenol level in cultured endothelial cells treated with LPC. CONCLUSION: GbE protected endothelial cells against LPC-induced damage due to reduction in lipid peroxidation and facilitation of synthesis and/or release of eposprostenol.
摘要:
Probucol (PBC) is an unique antiatherogenic drug producing its effect by antioxidant action rather than hypolipidaemic effect. However, the exact mechanism of its antiatherogenic effect is unclear. Therefore we investigated the PBC effects on the adhesion of monocytes to endothelial cells, an early event in atherogenesis. Monocyte adhesion to cultured pig aortic endothelial cells (EC) was induced by oxidized low density lipoprotein (Ox-LDL). To elucidate the mechanisms of the inhibition on adhesion, PBC effects on the Ox-LDL-induced expression of P-selectin, on the synthesis of von Willebrand factor (vWF) and prostacyclin (PGI(2)) were examined. The results showed that Ox-LDL enhanced the adhesion of monocytes to EC in a concentration-dependent and time-related manner. PBC 25, 50 and 75 mu mol/L inhibited the Ox-LDL-induced adhesion index from 37.3 % to 19.7, 16.6 and 14.6 % respectively (p all < 0.05), and inhibited the Ox-LDL-induced expression of P-selectin from 293.0 ng/ml to 180.0, 132.9 and 132.6 ng/ml respectively. Furthermore, PBC significantly attenuated the Ox-LDL-impaired synthesis of PGI(2) and vWF. These results indicate that PBC may provide a new approach in the prevention of atherosclerosis (AS) by intervention of monocyte adhesion to EC. In conclusion, PBC inhibits the Ox-LDL-induced adhesion of monocytes to EC. This effect is associated with the inhibition of the Ox-LDL-induced expression of P-selectin and the protection on the synthesis of PGI2.
作者机构:
[CHEN, X; LIAO, DF] HUNAN MED UNIV,DEPT PHARMACOL,CHANGSHA 410078,PEOPLES R CHINA.;[LIAO, DF] HENGYANG MED COLL,DEPT PHARMACOL,HENGYANG 421001,PEOPLES R CHINA.
通讯机构:
[LIAO, DF] H;HENGYANG MED COLL,DEPT PHARMACOL,HENGYANG 421001,PEOPLES R CHINA.
摘要:
We report here the protective effect of the angiotensin converting enzyme inhibitors captopril and ramiprilat against damage due to oxygen free radicals on aortic endothelium in a superfusion cascade system. Oxygen free radicals were generated by electrolysis of Krebs solution. Acetylcholine was infused through the donor aortic segment and relaxation of the detector aortic ring was used to indicate the release of endothelium-derived relaxing factor (EDRF). The percentage of relaxation before and after electrolysis was compared to calculate the relaxation index. Electrolyzed Krebs solution impaired the release of EDRF, as shown by a reduction in the relaxation index with a concomitant decrease in the tissue levels of superoxide dismutase and 6-keto PGF1 alpha and increase in malondialdehyde in the donor vessel. Captopril (100 microM), 15 microM ramiprilat and 30 nM iloprost had a protective effect as shown by a significantly smaller reduction in the relaxation index and the level of 6-keto PGF1 alpha and by an attenuation of the production of malondialdehyde. In addition, 1 microM indomethacin almost eliminated the protection by captopril. We conclude that both the SH-containing angiotensin-converting enzyme inhibitor captopril and the non-SH-containing ramiprilat, as well as iloprost, a stable analog of prostacyclin, can protect rabbit aortic endothelium against damage due to oxygen free radicals. The mechanism of such protection may be partly associated with the facilitation of the release of prostacyclin and consequent reduction in lipid peroxidation.