质粒超螺旋比例对其细胞转染效率及表达的影响

检测质粒开环、闭环比例不同时对其细胞转染效率及表达的影响。构建携带人肝细胞生长因子基因的质粒(pcDNA3-HGF)及携带LacZ报告基因的质粒(pcDNA3-LacZ),用核酸纯化试剂盒提取开环、闭环比例不同的质粒,然后用LipofectAMINE介导它们分别转染NIH3T3细胞,采用X-Gal染色和ELISA法分别观察转染效率和表达活性。结果表明,用超螺旋比例分别为85.45%和48.44%的质粒pcDNA3-LacZ转染生长旺盛的NIH3T3细胞,48h检测转染效率分别为23.4%±3.8%和9.3%±2.5%,前者约是后者的2.5倍。用超螺旋比例分别为93.28%和40.53%的质粒pcDNA3-HGF转染1×106NIH3T3细胞,ELISA法检测48h细胞培养上清中HGF的表达量分别为46.5±6.3ng和25.6±4.2ng,前者是后者的1.8倍。初步认为质粒开环、闭环比例不同对其细胞转染效率及表达有一定影响,超螺旋比例高时其转染效率及表达量较高。     

牛乳酪蛋白源抗菌肽的研究进展

抗菌肽是具有抗菌活性的肽类物质的总称。除了广泛存在于生物体内的内源性抗菌肽外,已从酪蛋白的酶解产物中获得了多种外源性抗菌肽。抗菌肽的抗菌机理独特,有望成为新一代抗菌剂。简要综述了牛乳酪蛋白源抗菌肽的种类、抗菌机理及其研究展望。     

海拔高度对2型糖尿病患者循环内皮祖细胞及低氧诱导因子的影响*

目的: 探讨不同海拔地区2型糖尿病患者循环内皮祖细胞数量及相关检测指标的变化情况,为2型糖尿病血管并发症的研究和治疗提供依据。方法: 选取386 m低海拔地区(咸阳市)和1 520 m高海拔地区(兰州市)各一家医院内被诊断的2型糖尿病患者(25人/29人)和健康体检者(20人/20人)。用全自动生化分析仪检测两组人群的血脂、血糖和糖化血红蛋白指标,用酶联免疫吸附试验(ELISA)检测低氧诱导因子-1α(HIF-1α)的浓度,用流式细胞仪测定外周血循环内皮祖细胞(EPCs)的数量。结果: 无论在低海拔还是高海拔地区,糖尿病组较健康组循环EPCs数量降低(P＜0.01),体质指数(BMI)、腰臀比(WHR)、甘油三酯(TG)、空腹血糖(FBG)及糖化血红蛋白含量(HbAlc)增高(P＜0.05);与低海拔组相比,无论高海拔的糖尿病患者还是健康者,HIF-1α的表达水平均明显增加(P＜0.05),而循环EPCs数量明显降低(P＜0.05),且有循环EPCs数量健康者高于2型糖尿病无血管并发症者高于2型糖尿病伴血管并发症者的表现(P＜0.05)。结论: 海拔高度增加,2型糖尿病(T2DM)患者体内HIF-1α表达水平增加,循环EPCs数量降低,且与其血管病变程度密切相关。因此有望通过对高海拔地区T2DM患者进行EPCs的移植来实现对糖尿病血管并发症的预防和改善。     

Population Genetics of the Washington National Primate Research Center's (WaNPRC) Captive Pigtailed Macaque (Macaca nemestrina) Population

Pigtailed macaques (Macaca nemestrina) provide an important model for biomedical research on human disease and for studying the evolution of primate behavior. The genetic structure of captive populations of pigtailed macaques is not as well described as that of captive rhesus (M. mulatta) or cynomolgus (M. fascicularis) macaques. The Washington National Primate Research Center houses the largest captive colony of pigtailed macaques located in several different housing facilities. Based on genotypes of 18 microsatellite (short tandem repeat [STR]) loci, these pigtailed macaques are more genetically diverse than captive rhesus macaques and exhibit relatively low levels of inbreeding. Colony genetic management facilitates the maintenance of genetic variability without compromising production goals of a breeding facility. The periodic introduction of new founders from specific sources to separate housing facilities at different times influenced the colony's genetic structure over time and space markedly but did not alter its genetic diversity significantly. Changes in genetic structure over time were predominantly due to the inclusion of animals from the Yerkes National Primate Research Center in the original colony and after 2005. Strategies to equalize founder representation in the colony have maximized the representation of the founders’ genomes in the extant population. Were exchange of animals among the facilities increased, further differentiation could be avoided. The use of highly differentiated animals may confound interpretations of phenotypic differences due to the inflation of the genetic contribution to phenotypic variance of heritable traits. Am. J. Primatol. 74:1017‐1027, 2012. © 2012 Wiley Periodicals, Inc.     

慢性心力衰竭患者血清Galectin-3、hs-cTnT、Cys C和PTX-3水平变化及临床意义

目的:研究慢性心力衰竭(CHF)患者血清半乳糖凝聚素-3(Galectin-3)、高敏肌钙蛋白-T(hs-cTnT)、胱抑素C(Cys C)和正五聚体蛋白-3(PTX-3)水平变化及临床意义。方法:选择2017年2月～2018年6月我院收治的CHF患者100例,按照美国心脏病协会(NYHA)心功能分级标准将其分成NYHAⅡ级组39例、NYHAⅢ级组33例、NYHAⅣ级组28例,根据随访1年患者预后情况将主要心脏不良事件患者记作预后不良组(n=27),其余记为预后优良组(n=73),另取同期于我院进行体检的健康者30例作为对照组。分别比较CHF患者和对照组的心功能指标、血清Galectin-3、hs-cTnT、Cys C、PTX-3水平,分析CHF患者上述指标的相关性及其与CHF预后情况的关系。结果:对照组、NYHAⅡ级组、NYHAⅢ级组、NYHAⅣ级组左心室舒张末期内径(LVEDD)呈逐渐增大趋势,而左心室射血分数(LVEF)呈逐渐降低趋势(P0.05)。对照组、NYHAⅡ级组、NYHAⅢ级组、NYHAⅣ级组血清Galectin-3、hs-cTnT、Cys C和PTX-3水平呈逐渐升高趋势(P0.05)。经Pearson相关性分析可得:CHF患者LVEDD与血清Galectin-3、hs-cTnT、Cys C、PTX-3水平呈正相关关系,而LVEF与血清Galectin-3、hs-cTnT、Cys C、PTX-3水平呈负相关关系(P0.05)。CHF预后优良组血清Galectin-3、hs-cTnT、Cys C、PTX-3水平均低于预后不良组(P0.05)。结论:CHF血清Galectin-3、hs-cTnT、Cys C和PTX-3水平均呈明显高表达,且与患者的病情严重程度呈密切相关,临床上可通过检测上述指标水平,继而为CHF临床诊断、预后评估提供参考依据。     

Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

Background  

Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis.     

MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system

Background  

MicroRNA (miRNA) encoding genes are abundant in vertebrate genomes but very few have been studied in any detail. Bioinformatic tools allow prediction of miRNA targets and this information coupled with knowledge of miRNA expression profiles facilitates formulation of hypotheses of miRNA function. Although the central nervous system (CNS) is a prominent site of miRNA expression, virtually nothing is known about the spatial and temporal expression profiles of miRNAs in the brain. To provide an overview of the breadth of miRNA expression in the CNS, we performed a comprehensive analysis of the neuroanatomical expression profiles of 38 abundant conserved miRNAs in developing and adult zebrafish brain.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H- heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.