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：
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：
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：
目的研究醛糖还原酶相似蛋白(aldose reductase like protein,ARL-1)在肝癌细胞中的功能及ARL-1基因高表达与肝癌耐药之间的关系.方法通过阳离子脂质体转染法将ARL-1基因导入HepG2细胞,建立稳定表达该基因的细胞株,采用MTT法检测细胞对含醛基药物的耐药变化情况.结果转染细胞与未转染细胞相比,对含醛基的抗肿瘤药物如阿霉素、丝裂霉素的耐药性明显增强,其半数抑制浓度(IC50)分别提高到2.6倍和3.1倍,ADM组t=6.39,P＜0.05,MMC组t=30.06,P＜0.01.用不含醛基的5-氟尿嘧啶处理两组细胞,则未发现有明显的耐药差异(t=0.684,P＞0.05).结论ARL-1的表达上调可能是肿瘤细胞对含醛基抗肿瘤药物耐药的主要原因之一.
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.
作者机构：
[唐圣松; 耿以琪; 陈桂彬; 饶青; 吴克复] State Key Laboratory of Experimental Hematology, Institute Of Hematology, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin, 300020, China
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.
通讯机构：
[Jiang, B.] C;Center of Molecular Biology, Hengyang Medical College, China
关键词：
群体遗传学;短串联重复序列;聚合酶链式反应
摘要：
目的:了解广州汉族人在vWA31A等6个短串联重复序列(short tandem repeat,STR)位点法医学的有关数据.方法:运用STR-PCR、4%变性聚丙烯酰胺凝胶电泳,结合荧光DNA自动检测技术,对汉族人群6个STR位点的等位基因频率和基因型频率进行了调查,并与其他种族或人群的等位基因频率进行了比较.结果:6个STR位点是vWA31A、TH01、F13A01、FES、TPOX和CSF1PO,所有位点的结果均符合Hardy-Weinberg平衡,且各位点等位基因间无相关性,除vWA31A外,其它5个位点的杂合度均低于白种人和黑种人.6个位点的累积DP=0.99999,PE=0.9708,pM=1.059×10-5.结论:所得到的等位基因频率等数据可为中国汉族人群法医个人识别、亲子鉴定及遗传学研究提供依据.
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.
