作者机构：
City Hope Natl Med Ctr, Duarte, CA 91010 USA.;Nanhua Univ, SNP Inst, Hengyang, Peoples R China.;Genomapping Inc, Tianjin, Peoples R China.;[Li, K] City Hope Natl Med Ctr, 1500 E Duarte Rd, Duarte, CA 91010 USA.
通讯机构：
[Li, K] C;City Hope Natl Med Ctr, 1500 E Duarte Rd, Duarte, CA 91010 USA.
摘要：
The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer-extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by exo(+) DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.
期刊：
American Heart Journal,2005年150(5):1039-1045 ISSN：0002-8703
通讯作者：
Fu, MD
作者机构：
[Fu, MD] Sichuan Univ, W China Med Ctr, Dept Biochem & Mol Biol, Apolipoprot Res Unit, Chengdu 610041, Sichuan, Peoples R China.;Hoist Grp Postdoctoral Work Stn, Chengdu, Sichuan, Peoples R China.;Nanhua Univ, Dept Biochem & Mol Biol, Hengyang, Hunan, Peoples R China.
通讯机构：
[Fu, MD] S;Sichuan Univ, W China Med Ctr, Dept Biochem & Mol Biol, Apolipoprot Res Unit, Chengdu 610041, Sichuan, Peoples R China.
摘要：
Background: To investigate the alterations of high-density lipoprotein (HDL) subclasses in endogenous hypertriglyceridemic subjects. Methods: Apolipoprotein A-I contents of plasma HDL subclasses were quantitated by 2-dimensional gel electrophoresis in 236 normolipidemic subjects (including 146 males and 90 females) and 176 endogenous hypertriglyceridemic subjects (including 103 males and 73 females). Results: Apolipoprotein A-I contents of small-sized pre-β1-HDL and HDL3a were significantly higher (P < .01 and P < .01, respectively), but those of large-sized HDL2a and HDL2b were significantly lower (P < .01 and P < .01, respectively) in hypertriglyceridemic subjects versus normolipidemic subjects. Moreover, with the elevation of triglyceride levels, small-sized pre-β1-HDL and HDL3a increased successively; however, large-sized HDL2a and HDL2b decreased successively. Males had significantly higher apolipoprotein A-I contents of small-sized pre-β1-HDL and HDL3b (P < .05 and P < .05, respectively), but lower contents of large-sized HDL2b (P < .01) than females in both normolipidemic and hypertriglyceridemic subjects. Conclusions: The particle size of HDL shifted toward smaller size in hypertriglyceridemic subjects, especially in male subjects. Of note, the shift was more obvious with the elevation of triglyceride levels. The changes mentioned above indicate that HDL maturation might be abnormal and reverse cholesterol transport might be weakened.
摘要：
To investigate the inhibitory effect of the Bcl-XL small interfering RNA (siRNA) on Bcl-XL gene expression in the human gastric cancer cell line MGC-803, green fluorescent protein (GFP) siRNA was constructed and transfected into MGC-803 cells, together with GFP expression vector pTrace SV40. GFP expression levels were observed using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected by acridine orange (AO) and flow cytometry. Results were as follows: (1) 48 h after GFP expression vector and GFP siRNA co-transfection, the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group, according to fluorescence microscopy results. The mRNA and protein levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced to almost background level compared with negative siRNA transfectants or untreated cells. (2) Changes in nucleus morphology was observed by AO staining nucleic and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants have an increased spontaneous apoptosis (21.17%±1.26% vs. 1.19%±0.18% and 1.56%±0.15% respectively, P<0.05 vs. negative siRNA or untreated control). siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or Bcl-XL expression in MGC-803 cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may be a beneficial agent against human gastric adenocarcinoma.
摘要：
Type 2 diabetes is a major risk factor of the development of atherosclerosis in humans. However, studies examining mechanisms underlying diabetes-accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. Pigs have a cardiovascular system that is very similar to that of humans and is useful as a model for human physiology and pathophysiology. In this study, we established a new miniature pig model for studying dyslipidaemia and atherosclerosis in diabetes. Chinese Guizhou minipigs were fed a normal control diet or a high-fat/high-sucrose diet (HFSD) for 6 months. Plasma total cholesterol (TC), high-density lipoprotein cholesterol, triglyceride (TG), insulin and glucose were quantified at monthly intervals. The induction of insulin resistance and dysfunction of the pancreatic β-cell were assessed by oral glucose tolerance test and insulin sensitivity test. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. During the feeding period, mild high plasma TC and TG were induced. At the end of 6 months, in HFSD-fed animals, the adipocytes were hypertrophic, fat deposit in the liver was observed, loss of pancreatic β-cells was observed, and the aortic fatty streak lesions were clearly present in the animals' aortas. Our study established that miniature pigs that were fed a HFSD without adding dietary cholesterol developed insulin resistance, mild diabetes and atherosclerotic lesions. HFSD-fed miniature pigs may be good animal models for research on the treatment of diabetic dyslipidaemia complicated with atherosclerosis.
期刊：
JOURNAL OF ENDOCRINOLOGY,2004年180(3):399-408 ISSN：0022-0795
通讯作者：
Yin, W
作者机构：
[Yin, W] Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Mol Biol, Hunan 421001, Peoples R China.;Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Biotechnol, Hunan 421001, Peoples R China.;Cent S Univ, Xiangya Med Coll, Dept Pathophysiol, Changsha, Hunan, Peoples R China.;Otsuka Pharmaceut Fact Inc, Res & Dev, Tokushima, Japan.;Univ Tsukuba, Inst Basic Med Sci, Dept Pathol, Lab Cardiovasc Dis, Tsukuba, Ibaraki 3058575, Japan.
通讯机构：
[Yin, W] N;Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Mol Biol, Hunan 421001, Peoples R China.
摘要：
The synthetic compound NO-1886 (ibrolipim) is a lipoprotein lipase activator that has been proven to be highly effective in lowering plasma triglycerides. Recently, we found that NO-1886 also reduced plasma free fatty acids and glucose in high-fat/high-sucrose diet-induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 treatment on ectopic lipid deposition and the islet pathology in miniature swine fed a high-fat/high-sucrose diet. Our results showed that feeding this diet to miniature swine caused insulin resistance, increased lipid deposition in non-adipose tissue, such as in the heart, skeletal muscle, liver and pancreas, and also caused pancreatic beta cell damage. However, supplementing 1% NO-1886 (200 mg/kg per day) into the high-fat/high-sucrose diet decreased ectopic lipid deposition, improved insulin resistance, and alleviated the beta cell damage. These results suggest that improvement of lipid disorder, non-adipose tissue steatosis and insulin resistance may be very important for the protection of beta cell damage. Therefore, NO-1886 is potentially beneficial for the treatment of insulin-resistance syndrome.
摘要：
Background. This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans. Methods. Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor κB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting. Results. M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression. Conclusion. This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.
摘要：
DNA polymerases without the 3' exonuclease function ($exo^-$ pol) have been widely used in sequencing and SNP genotyping. As a major player that expedited the coming of the postgenomic era, $exo^-$ polymerases worked remarkably well in the Human Genome Sequencing Project. However, it has become a challenge for this class of polymerases to efficiently screen the large number of SNPs that are found in the human genome. For more than three decades it has been recognized that polymerase fidelity varied according to the presence of proofreading activity that is mediated by its internal 3' exonuclease. Polymerases with proofreading function are famous for their high fidelity in DNA replication both in vivo and in vitro, but this well-known class of polymerases has been almost completely neglected in genetic analysis in the postgenomic era. We speculate that $exo^+$ polymerases may exhibit higher nucleotide identification ability when compared to $exo^-$ polymerases for an in vitro genetic analysis. With the application of $exo^+$ polymerases in SNP assays, a novel mechanism for the maintenance of DNA replication, the on/off switch, was discovered. Two new SNP assays have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of $exo^+$ polymerases. Furthermore, the on/off switch mechanism embodies a powerful nucleotide identification ability, which can be used to discriminate the bases that are upstream of the 3' terminus, and thus defines a new concept in de novo sequencing technology. Application of $exo^+$ polymerases to genetic analysis, and especially SNP assays, will greatly accelerate the pace to personalized medicine.
作者机构：
[Yang, J. H.; Yang, Y. Z.; Tang, C. K.; Yil, G. H.; Yan, Wan; Mo, Z. C.] Nanhua Univ, Inst Cardiovasc Res, Hengyang, Peoples R China.;[Xi, S. M.; Wang, Z. B.; Yin, W. D.] Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Mol Biol, Hengyang, Peoples R China.
作者机构：
[Li, K] Genomapping Inc, Tianjin, Peoples R China.;Jinan Univ, Inst Life Sci & Biotechnol, Guangzhou, Peoples R China.;Chugai Pharma USA, San Diego, CA USA.;Nanhua Univ, Inst Pharm & Pharmacol, Hengyang, Peoples R China.
通讯机构：
[Li, K] G;Genomapping Inc, Tianjin, Peoples R China.
关键词：
exo plus polymerase;proofreading;SNP;3 ' terminal-labeled primer;phosphorothioate
期刊：
DRUGS OF THE FUTURE,2004年29(1):53-62 ISSN：0377-8282
通讯作者：
Yin, W
作者机构：
[Yin, W] Nanhua Univ, Dept Biochem & Mol Biol, Sch Life Sci & Technol, Hengyang 421001, Peoples R China.;Nanhua Univ, Cardiovasc Res Inst, Sch Med, Hengyang 421001, Peoples R China.;Otsuka Pharmaceut Factory Inc, Res & Dev, Naruto, Tokushima 7728601, Japan.
通讯机构：
[Yin, W] N;Nanhua Univ, Dept Biochem & Mol Biol, Sch Life Sci & Technol, Hengyang 421001, Peoples R China.
摘要：
Lipoprotein lipase (LPL) is a rate-limiting enzyme that hydrolyzes circulating triglyceride (TG)-rich lipoproteins such as very low-density lipoproteins (VLDL) and chylomicrons. A decrease in LPL activity is associated with an increase in plasma 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 (CVD). Tsutsumi et al. hypothesized that elevating LPL activity would cause a reduction in plasma TG and an increase in plasma HDL-C, resulting in protection against the development of atherosclerosis. To test this hypothesis, Otsuka synthesized the LPL activator NO-1886. The effects of NO-1886 in animals have been extensively studied. NO-1886 has been shown to increase LPL mRNA and LPL activity in adipose tissue, myocardium and skeletal muscle, resulting in an elevation of post-heparin plasma LPL activity and LPL mass in rats. NO-1886 has also been shown to decrease plasma TG concentration and to cause 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 aorta. The results of multiple regression analysis in these studies suggested that the increase in plasma HDL-C and the decrease in plasma TG protected against atherosclerosis. These results show that the atherogenic lipid profile is changed to an antiatherogenic lipid profile by increasing LPL activity, resulting in protection against the development of atherosclerosis. Therefore, the LPL activator NO-1886 is potentially beneficial for the treatment of hypertriglyceridemia and hypo-HDL-cholesterolemia, and for protection against atherosclerosis. Furthermore, we hypothesized that elevation of LPL activity in adipose tissue would cause an improvement in cachexia, and elevation of LPL activity in skeletal muscle would lead to an improvement in obesity, because the LPL in adipose tissue is related to fat storage and LPL in skeletal muscle is related to free fatty acid (FFA) oxidation. From the many published studies, we confirmed that NO-1886 improved cachexia by elevating LPL activity in adipose tissue and improved obesity by elevating LPL activity in skeletal muscle. It is concluded that NO-1886, and possibly other LPL-activating agents, protect against atherosclerosis, as well as cachexia and obesity.
摘要：
The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.
作者机构：
[Jia Zhang; Zhang, X; Chen, LL; Guo, ZF; Peng, CY] Genomapping Inc., Tianjin;[Meng, B; Zhou, LY; Hu, YH] Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai;[Liao, DF; Zhu, BY] Institute of SNP, Institute of Pharmacy and Pharmacology, National University, Hengyang;[Lee, PP] University of California in San Diego, San Diego, USA;[Xu, XM] Department of Medical Genetics, The First Military Medical University, Guangzhou
通讯机构：
[Li, K ] ;Genomapping Inc, Tianjin, Peoples R China.
关键词：
gene synthesis;SARS;PCR;sequential primer extension
摘要：
A novel coronavirus was identified as the cause for severe acute respiratory syndrome (SARS). The complete sequence of SARS genome has provided an opportunity for the development of molecular diagnostic assays. To restrain further outbreak of SARS, the World Health Organization has posted several pairs of polymerase chain reaction (PCR) primers for early diagnosis and urged more research to be done on PCR protocols. Here we report a strategy for the de novo synthesis of PCR templates complimentary to the SARS virus genome, which has the advantage of working on PCR templates without concern about viral infection and also has the advantage that it can be used by those who do not have access to the SARS virus. This highly efficient and safe strategy for obtaining SARS gene fragments is useful for the development of PCR assays, as well as for the preparation of reliable positive controls for PCR testing kits.
摘要：
With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3′-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo+ polymerases was further evaluated in primer extension using three types of 3′ terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3′ exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3′-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.
